Branched Peg Remodeling and Glycosylation of Glucagon-Like Peptides-1 [Glp-1]

ABSTRACT

The present invention provides polypeptides that include an O-linked glycoconjugate in which a species such as a water-soluble polymer, a therapeutic agent of a biomolecule is covalently linked through an intact O-linked glycosyl residue to the polypeptide. The polypeptides of the invention include wild-type peptides and mutant peptides that include an O-linked glycosylation site that is not present in the wild-type peptide. Also provided are methods of making the peptides of the invention and methods, pharmaceutical compositions containing the peptides and methods of treating, ameliorating or preventing diseased in mammals by administering an amount of a peptide of the invention sufficient to achieve the desired response.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a U.S. national phase application ofPCT/US2005/024781 filed Jul. 13, 2005 and claims priority to U.S.Provisional Patent Application No. 60/587,738, filed Jul. 13, 2004 andU.S. Provisional Patent Application No. 60/608,723 filed Sep. 10, 2004,the disclosures of which are incorporated herein by reference in theirentirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to O-linked glycosylated glycopeptides,particularly glucagon-like peptide-1 (GLP-1) and GLP-1 peptide mutantsthat include O-linked glycosylation sites not present in the wild-typepeptide.

BACKGROUND OF THE INVENTION

Glucagon-like peptide-1 (GLP-1) is an important glucoincretin hormonesecreted from intestinal L cells in response to nutrient ingestion.GLP-1 functions to regulate plasma glucose levels via variousindependent mechanisms, making it an ideal candidate for treatment ofdiabetes, and possibly useful in the pharmacotherapy of obesity.

The biologically active forms of GLP-1 possess multiple functions invivo, including enhancement of glucose-dependent insulin secretion,stimulation of proinsulin gene expression, and supression of glucagonsecretion and gastric emptying. GLP-1 also enhances insulin sensitivity,induces β cell differentiation and proliferation, decreases caloricintake, and increases satiety.

The mature, active form of GLP-1 is a 30 amino acid derivative ofproglucagon, a 160 amino acid prohormone. GLP-1 is synthesized by posttranslational processing of proglucagon in intestinal L cells.Postranslational processing of proglucagon gives rise to glucagon,GLP-1, GLP-2 and other peptide sequences, IP-1 and IP-2, of unknownfunction. The initial GLP-1 cleaved from proglucagon is furtherprocessed first by N-terminal cleaveage to form a biologically activepeptide (GLP-1₍₇₋₃₇₎). GLP-1₍₇₋₃₇₎ is then C-terminally truncated andamidated to form the predominant biolgically active species,GLP-1_((7-36)amide).

GLP-1_((7-36)amide) has a very short half life in vivo. The plasma halflife of GLP-1 is about 5 minutes, and the metabolic clearance rate isabout 12-13 minutes. In circulation, the predominant form of GLP-1 israpidly inactivated as a result of degradation by dipeptidyl-peptidaseIV (see e.g., Deacon et al. (1995) Endocrinol. Metab. 80:952-957, andHansen et al. (1999) Endocrinology 140:5356). GLP-1_((7-36)amide) isalso susceptible to degradation by neutral endopeptidases, including NEP24.11 (Sodman et al. (1995) Reg. Peptides 58:149-156).

The unique ability of GLP-1 to lower postprandial hyperglycemia viathree independent and complementary mechanisms of action (increasedinsulin secretion, inhibition of glucagon release, and inhibition ofgastrointestinal motility) are what make this peptide hormone an idealcandidate for the treatment of diabetes. Indeed, GLP-1 providesunprecedented advantages over any other pharmacological agent currentlyavailable. Unfortunately, despite its potential, there are seriouslimitations to the possible therapeutic use of GLP-1 in humans. The mostserious limitation is the very short half life of GLP-1 in vivo. Evenwhen administered subcutaneously, peak concentrations return to baselinewithin 90 minutes.

The therapeutic potential of GLP-1 and its very short half life haveprompted the search for and discovery of analogs that may provide anextended GLP-1-like biological activity. Several analogs have beenisolated from other species (Fehmann, H. C., et al. (1995) EndocrineReviews 16:390-410, and Thorens B. et al. (1993) Diabetes 42:1678-1682),and mutant GLP-1 peptides resistant to degradation have been created(Xiao et al. (2001) Biochemistry 40:2860-2869).

Some GLP-1 analogs may show some promise as therapeutics. However, sinceGLP-1 peptide is a highly multifunctional protein, mutants andinterspecies homologs may have unpredictable plieotropic effects.Indeed, Xiao et al. showed that some mutants exhibit altered biologicalactivity independent of any changes in receptor binding activity. Thus,the biological activities of GLP-1 can be uncoupled from one another.

Diabetes, obesity and other disorders of sugar metabolism and glycemiccontrol carry a very high price for the individual, as well as thesociety in terms of health, lost productivity and the loss of wages andfinancial output. Thus, there is clearly a need in the art for effectivemedications that facilitate glycemic control in the individual. Astabilized GLP-1 with increased half life in vivo could meet this need.Preferably a stabilized GLP-1 peptide would be very similar to the wildtype protein, such that changes to biological activity, and hencepossible side effects of therapy can be minimized. The present inventionanswers the need for stabilized GLP-1 molecules, thereby providingtherapeutically effective GLP-1 peptides. Other objects and advantageswill become apparent from the detailed description that follows.

BRIEF SUMMARY OF THE INVENTION

Diabetes and disorders of glycemic control are serious conditions which,if unchecked can have dire consequences for the individual and societyat large. Although type 1 diabetes can be controlled more or lesseffectively with insulin injections, there are multiple pathways ofglycemic control. If some of those pathway could also be recruited intotherapeutic methods, glycemic control for diabetics would be improved.Further, enhanced glycemic control for type 2 diabetics and individualsstruggling with obesity, could provide enhanced health benefits forthese groups of individuals as well.

As noted above, Glucagon-Like Peptide-1 (GLP-1) facilitates glycemiccontrol in the individual by multiple mechanisms. Thus, GLP-1 is anideal candidate for the pharmacotherapy of glycemic disorders.Unfortunately, the potential therapeutic uses of GLP-1 are limited bythe short in vivo half life of the protein. Fortunately, methods thatimprove in vivo half life of the protein have now been discovered. Thesemethods have the added advantage that they introduce minimal alterationsto the protein and therefore the risks of side effects are minimized.

Indeed, it has now been discovered that enzymatic glycoconjugationreactions can be specifically targeted to O-linked glycosylation sitesand to glycosyl residues that are attached to O-linked glycosylationsites. The targeted O-linked glycosylation sites can be sites native toa wild-type peptide or, alternatively, they can be introduced into apeptide by mutation. Thus, a method for prolonging the in vivo half lifeof GLP-1 (and other proteins) is provided by the methods of theinvention.

In addition to the discovery that O-linked glycosylation sites, andglycosyl residues linked thereto, are useful targets forglycoconjugation reactions, the present invention provides mutantpolypeptides in which the amino acid sequence is manipulated by mutationto insert, remove or relocate one or more O-linked glycosylation site inthe peptide. When a site is added or relocated, it is not present or notpresent in a selected location in the wild type peptide. The mutantO-linked glycosylation site is a point of attachment for a modifiedglycosyl residue that is enzymatically conjugated to the O-linkedglycosylation site. Using the methods of the invention, theglycosylation site can be shifted to any efficacious position on thepeptide. For example, if the native glycosylation site is sufficientlyproximate the active site of the peptide that conjugation of a largewater-soluble polymer interferes with the biological activity of thepeptide, it is within the scope of the invention to engineer a mutantpeptide that includes an O-linked glycosylation site as removed from theactive site as necessary to provide a biologically active peptideconjugate.

Post-expression in vitro modification of peptides is an attractivestrategy to remedy the deficiencies of methods that rely on controllingglycosylation by engineering expression systems; including bothmodification of glycan structures or introduction of glycans at novelsites. A comprehensive toolbox of recombinant eukaryoticglycosyltransferases is becoming available, making in vitro enzymaticsynthesis of mammalian glycoconjugates with custom designedglycosylation patterns and glycosyl structures possible. See, forexample, U.S. Pat. Nos. 5,876,980; 6,030,815; 5,728,554; 5,922,577; andWO/9831826; US2003180835; and WO 03/031464.

In vitro glycosylation offers a number of advantages compared torecombinant expression of glycoproteins of which custom design andhigher degree of homogeneity of the glycosyl moiety are examples.Moreover, combining bacterial expression of glycotherapeutics with invitro modification (or placement) of the glycosyl residue offersnumerous advantages over traditional recombinant expression technologyincluding reduced potential exposure to adventitious agents, increasedhomogeneity of product, and cost reduction.

In addition to methods of O-linked glycosylation, inserting O-linkedglycosylation sites into peptides and methods of glycosylating theinserted sites, the present invention provides methods of improvingpharmacological parameters of glycopeptide therapeutics, e.g., alteringpharmacokinetics, pharmacodynamics and bioavailability of therapeutic(glyco)proteins, e.g., hormones, and enzymes. In particular, theinvention provides a method for lengthening the in vivo half-lives ofglycopeptide therapeutics by conjugating a water-soluble polymer to thepeptide through an intact glycosyl linking group. In an exemplaryembodiment, covalent attachment of polymers, such as polyethylene glycol(PEG), e.g, m-PEG, to such peptides affords conjugates having in vivoresidence times, and pharmacokinetic and pharmacodynamic properties,enhanced relative to the unconjugated peptide.

Art-recognized methods of covalent PEGylation rely on chemicalconjugation through reactive groups on amino acids or carbohydrates. Amajor shortcoming of chemical conjugation of PEG to proteins orglycoproteins is lack of selectivity, which often results in attachmentof PEG at sites implicated in protein or glycoprotein bioactivity.Several strategies have been developed to address site selectiveconjugation chemistries, however, one universal method suitable for avariety of recombinant proteins has yet to be developed.

In contrast to art-recognized methods, the present invention provides anovel strategy for highly selective site directed O-linkedglycoconjugation, e.g., glyco-PEGylation. In an exemplary embodiment ofthe invention, site directed attachment sites for PEGylation areprovided by in vitro enzymatic GalNAc O-linked glycosylation of specificpeptide sequences, e.g., mutant sequences, containing serine andthreonine residues. The recombinant proteins are preferably expressed inbacteria, e.g., E. coli, to avoid host cell glycosylation.Glyco-PEGylation is subsequently performed enzymatically utilizing aglycosyltransferase, e.g., a sialyltransferase, capable of transferringthe species PEG-glycosyl, e.g., PEG-sialic acid, to an O-linkedglycosylation site (“glyco-PEGylation”). O-linked glycosylation sitesmay be introduced into any peptide sequence by providing a mutantpeptide with simple short sequence motifs.

Additional aspects, advantages and objects of the present invention willbe apparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a list of peptides which can be used as substrates forthis invention.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations

PEG, poly(ethyleneglycol); m-PEG, methoxy-poly(ethylene glycol); PPG,poly(propyleneglycol); m-PPG, methoxy-poly(propylene glycol); Fuc,fucosyl; Gal, galactosyl; GalNAc, N-acetylgalactosaminyl; Glc, glucosyl;GlcNAc, N-acetylglucosaminyl; Man, mannosyl; ManAc, mannosaminylacetate; Sia, sialic acid; and NeuAc, N-acetylneuraminyl.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization are those well known and commonly employedin the art. Standard techniques are used for nucleic acid and peptidesynthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well known and commonlyemployed in the art. Standard techniques, or modifications thereof, areused for chemical syntheses and chemical analyses.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, and mRNA encoded by a gene.

The term “gene” means the segment of DNA involved in producing apolypeptide chain. It may include regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames that flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds having a structure that is different from the generalchemical structure of an amino acid, but that functions in a mannersimilar to a naturally occurring amino acid.

There are various known methods in the art that permit the incorporationof an unnatural amino acid derivative or analog into a polypeptide chainin a site-specific manner, see, e.g., WO 02/086075.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein that encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

In the present application, amino acid residues are numbered accordingto their relative positions from the left most residue, which isnumbered 1, in an unmodified wild-type polypeptide sequence.

“Peptide” refers to a polymer in which the monomers are amino acids andare joined together through amide bonds, alternatively referred to as apolypeptide. Additionally, unnatural amino acids, for example,β-alanine, phenylglycine and homoarginine are also included. Amino acidsthat are not gene-encoded may also be used in the present invention.Furthermore, amino acids that have been modified to include reactivegroups, glycosylation sites, polymers, therapeutic moieties,biomolecules and the like may also be used in the invention. All of theamino acids used in the present invention may be either the D- orL-isomer. The L-isomer is generally preferred. In addition, otherpeptidomimetics are also useful in the present invention. As usedherein, “peptide” refers to both glycosylated and unglycosylatedpeptides. Also included are peptides that are incompletely glycosylatedby a system that expresses the peptide. For a general review, see,Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDESAND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267(1983).

In the present application, amino acid residues are numbered accordingto their relative positions from the left most residue, which isnumbered 1, in a peptide sequence.

“Proximate a proline residue,” as used herein refers to an amino acidthat is less than about 10 amino acids removed from a proline residue,preferably, less than about 9, 8, 7, 6 or 5 amino acids removed from aproline residue, more preferably, less than about 4, 3, 2 or 1 residuesremoved from a proline residue. The amino acid “proximate a prolineresidue” may be on the C- or N-terminal side of the proline residue.

The term “sialic acid” refers to any member of a family of nine-carboncarboxylated sugars. The most common member of the sialic acid family isN-acetyl-neuraminic acid(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265:21811-21819 (1990)). Also included are 9-substituted sialic acids suchas a 9-O—C₁-C₆ acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);Sialic Acids. Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992)). The synthesis and use of sialic acidcompounds in a sialylation procedure is disclosed in internationalapplication WO 92/16640, published Oct. 1, 1992.

As used herein, the term “modified sugar,” refers to a naturally- ornon-naturally-occurring carbohydrate that is enzymatically added onto anamino acid or a glycosyl residue of a peptide in a process of theinvention. The modified sugar is selected from a number of enzymesubstrates including, but not limited to sugar nucleotides (mono-, di-,and tri-phosphates), activated sugars (e.g., glycosyl halides, glycosylmesylates) and sugars that are neither activated nor nucleotides. The“modified sugar” is covalently functionalized with a “modifying group.”Useful modifying groups include, but are not limited to, water-solublepolymers, therapeutic moieties, diagnostic moieties, biomolecules andthe like. The modifying group is preferably not a naturally occurring,or an unmodified carbohydrate. The locus of functionalization with themodifying group is selected such that it does not prevent the “modifiedsugar” from being added enzymatically to a peptide.

The term “water-soluble” refers to moieties that have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like. Peptides can have mixed sequencesof be composed of a single amino acid, e.g., poly(lysine). An exemplarypolysaccharide is poly(sialic acid). An exemplary poly(ether) ispoly(ethylene glycol), e.g., m-PEG. Poly(ethylene imine) is an exemplarypolyamine, and poly(acrylic) acid is a representative poly(carboxylicacid).

The polymer backbone of the water-soluble polymer can be poly(ethyleneglycol) (i.e. PEG). However, it should be understood that other relatedpolymers are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended to beinclusive and not exclusive in this respect. The term PEG includespoly(ethylene glycol) in any of its forms, including alkoxy PEG,difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(-PEG-OH).sub.m in which R represents the core moiety,such as glycerol or pentaerythritol, and m represents the number ofarms. Multi-armed PEG molecules, such as those described in U.S. Pat.No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as poly(propylene glycol) (“PPG”), copolymers of ethyleneglycol and propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxypropylmethacrylamide), poly(α-hydroxy acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),such as described in U.S. Pat. No. 5,629,384, which is incorporated byreference herein in its entirety, and copolymers, terpolymers, andmixtures thereof. Although the molecular weight of each chain of thepolymer backbone can vary, it is typically in the range of from about100 Da to about 100,000 Da, often from about 6,000 Da to about 80,000Da.

The term “glycoconjugation,” as used herein, refers to the enzymaticallymediated conjugation of a modified sugar species to an amino acid orglycosyl residue of a polypeptide, e.g., a mutant human growth hormoneof the present invention. A subgenus of “glycoconjugation” is“glycol-PEGylation,” in which the modifying group of the modified sugaris poly(ethylene glycol), and alkyl derivative (e.g., m-PEG) or reactivederivative (e.g., H2N-PEG, HOOC-PEG) thereof.

The terms “large-scale” and “industrial-scale” are used interchangeablyand refer to a reaction cycle that produces at least about 250 mg,preferably at least about 500 mg, and more preferably at least about 1gram of glycoconjugate at the completion of a single reaction cycle.

The term, “glycosyl linking group,” as used herein refers to a glycosylresidue to which a modifying group (e.g., PEG moiety, therapeuticmoiety, biomolecule) is covalently attached; the glycosyl linking groupjoins the modifying group to the remainder of the conjugate. In themethods of the invention, the “glycosyl linking group” becomescovalently attached to a glycosylated or unglycosylated peptide, therebylinking the agent to an amino acid and/or glycosyl residue on thepeptide. A “glycosyl linking group” is generally derived from a“modified sugar” by the enzymatic attachment of the “modified sugar” toan amino acid and/or glycosyl residue of the peptide. The glycosyllinking group can be a saccharide-derived structure that is degradedduring formation of modifying group-modified sugar cassette (e.g.,oxidation→Schiff base formation→reduction), or the glycosyl linkinggroup may be intact. An “intact glycosyl linking group” refers to alinking group that is derived from a glycosyl moiety in which thesaccharide monomer that links the modifying group and to the remainderof the conjugate is not degraded, e.g., oxidized, e.g., by sodiummetaperiodate. “Intact glycosyl linking groups” of the invention may bederived from a naturally occurring oligosaccharide by addition ofglycosyl unit(s) or removal of one or more glycosyl unit from a parentsaccharide structure.

The term “targeting moiety,” as used herein, refers to species that willselectively localize in a particular tissue or region of the body. Thelocalization is mediated by specific recognition of moleculardeterminants, molecular size of the targeting agent or conjugate, ionicinteractions, hydrophobic interactions and the like. Other mechanisms oftargeting an agent to a particular tissue or region are known to thoseof skill in the art. Exemplary targeting moieties include antibodies,antibody fragments, transferrin, HS-glycoprotein, coagulation factors,serum proteins, β-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO and the like.

As used herein, “therapeutic moiety” means any agent useful for therapyincluding, but not limited to, antibiotics, anti-inflammatory agents,anti-tumor drugs, cytotoxins, and radioactive agents. “Therapeuticmoiety” includes prodrugs of bioactive agents, constructs in which morethan one therapeutic moiety is bound to a carrier, e.g, multivalentagents. Therapeutic moiety also includes proteins and constructs thatinclude proteins. Exemplary proteins include, but are not limited to,Glucagon like protein-1 (GLP-1), Erythropoietin (EPO), GranulocyteColony Stimulating Factor (GCSF), Granulocyte Macrophage ColonyStimulating Factor (GMCSF), Interferon (e.g., Interferon-α, -β, -γ),Interleukin (e.g., Interleukin II), serum proteins (e.g., Factors VII,VIIa, VIII, IX, and X), Human Chorionic Gonadotropin (HCG), FollicleStimulating Hormone (FSH) and Lutenizing Hormone (LH) and antibodyfusion proteins (e.g. Tumor Necrosis Factor Receptor ((TNFR)/Fc domainfusion protein)).

As used herein, “anti-tumor drug” means any agent useful to combatcancer including, but not limited to, cytotoxins and agents such asantimetabolites, alkylating agents, anthracyclines, antibiotics,antimitotic agents, procarbazine, hydroxyurea, asparaginase,corticosteroids, interferons and radioactive agents. Also encompassedwithin the scope of the term “anti-tumor drug,” are conjugates ofpeptides with anti-tumor activity, e.g. TNF-α. Conjugates include, butare not limited to those formed between a therapeutic protein and aglycoprotein of the invention. A representative conjugate is that formedbetween PSGL-1 and TNF-α.

As used herein, “a cytotoxin or cytotoxic agent” means any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracinedione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Other toxinsinclude, for example, ricin, CC-1065 and analogues, the duocarmycins.Still other toxins include diptheria toxin, and snake venom (e.g., cobravenom).

As used herein, “a radioactive agent” includes any radioisotope that iseffective in diagnosing or destroying a tumor. Examples include, but arenot limited to, indium-111, cobalt-60. Additionally, naturally occurringradioactive elements such as uranium, radium, and thorium, whichtypically represent mixtures of radioisotopes, are suitable examples ofa radioactive agent. The metal ions are typically chelated with anorganic chelating moiety.

Many useful chelating groups, crown ethers, cryptands and the like areknown in the art and can be incorporated into the compounds of theinvention (e.g., EDTA, DTPA, DOTA, NTA, HDTA, etc. and their phosphonateanalogs such as DTPP, EDTP, HDTP, NTP, etc). See, for example, Pitt etal., “The Design of Chelating Agents for the Treatment of IronOverload,” In, INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE; Martell,Ed.; American Chemical Society, Washington, D.C., 1980, pp. 279-312;Lindoy, THE CHEMISTRY OF MACROCYCLIC LIGAND COMPLEXES; CambridgeUniversity Press, Cambridge, 1989; Dugas, BIOORGANIC CHEMISTRY;Springer-Verlag, New York, 1989, and references contained therein.

Additionally, a manifold of routes allowing the attachment of chelatingagents, crown ethers and cyclodextrins to other molecules is availableto those of skill in the art. See, for example, Meares et al.,“Properties of In Vivo Chelate-Tagged Proteins and Polypeptides.” In,MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICALASPECTS;” Feeney, et al., Eds., American Chemical Society, Washington,D.C., 1982, pp. 370-387; Kasina et al., Bioconjugate Chem., 9: 108-117(1998); Song et al., Bioconjugate Chem., 8: 249-255 (1997).

As used herein, “pharmaceutically acceptable carrier” includes anymaterial, which when combined with the conjugate retains the conjugates'activity and is non-reactive with the subject's immune systems. Examplesinclude, but are not limited to, any of the standard pharmaceuticalcarriers such as a phosphate buffered saline solution, water, emulsionssuch as oil/water emulsion, and various types of wetting agents. Othercarriers may also include sterile solutions, tablets including coatedtablets and capsules. Typically such carriers contain excipients such asstarch, milk, sugar, certain types of clay, gelatin, stearic acid orsalts thereof, magnesium or calcium stearate, talc, vegetable fats oroils, gums, glycols, or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients. Compositionscomprising such carriers are formulated by well known conventionalmethods.

As used herein, “administering” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, or subcutaneousadministration, administration by inhalation, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to the subject.Administration is by any route including parenteral and transmucosal(e.g., oral, nasal, vaginal, rectal, or transdermal), particularly byinhalation. Parenteral administration includes, e.g., intravenous,intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial. Moreover, whereinjection is to treat a tumor, e.g., induce apoptosis, administrationmay be directly to the tumor and/or into tissues surrounding the tumor.Other modes of delivery include, but are not limited to, the use ofliposomal formulations, intravenous infusion, transdermal patches, etc.

The term “isolated” refers to a material that is substantially oressentially free from components, which are used to produce thematerial. For peptide conjugates of the invention, the term “isolated”refers to material that is substantially or essentially free fromcomponents, which normally accompany the material in the mixture used toprepare the peptide conjugate. “Isolated” and “pure” are usedinterchangeably. Typically, isolated peptide conjugates of the inventionhave a level of purity preferably expressed as a range. The lower end ofthe range of purity for the peptide conjugates is about 60%, about 70%or about 80% and the upper end of the range of purity is about 70%,about 80%, about 90% or more than about 90%.

When the peptide conjugates are more than about 90% pure, their puritiesare also preferably expressed as a range. The lower end of the range ofpurity is about 90%, about 92%, about 94%, about 96% or about 98%. Theupper end of the range of purity is about 92%, about 94%, about 96%,about 98% or about 100% purity.

Purity is determined by any art-recognized method of analysis (e.g.,band intensity on a silver stained gel, polyacrylamide gelelectrophoresis, HPLC, or a similar means).

“Essentially each member of the population,” as used herein, describes acharacteristic of a population of peptide conjugates of the invention inwhich a selected percentage of the modified sugars added to a peptideare added to multiple, identical acceptor sites on the peptide.“Essentially each member of the population” speaks to the “homogeneity”of the sites on the peptide conjugated to a modified sugar and refers toconjugates of the invention, which are at least about 80%, preferably atleast about 90% and more preferably at least about 95% homogenous.

“Homogeneity,” refers to the structural consistency across a populationof acceptor moieties to which the modified sugars are conjugated. Thus,in a peptide conjugate of the invention in which each modified sugarmoiety is conjugated to an acceptor site having the same structure asthe acceptor site to which every other modified sugar is conjugated, thepeptide conjugate is said to be about 100% homogeneous. Homogeneity istypically expressed as a range. The lower end of the range ofhomogeneity for the peptide conjugates is about 60%, about 70% or about80% and the upper end of the range of purity is about 70%, about 80%,about 90% or more than about 90%.

When the peptide conjugates are more than or equal to about 90%homogeneous, their homogeneity is also preferably expressed as a range.The lower end of the range of homogeneity is about 90%, about 92%, about94%, about 96% or about 98%. The upper end of the range of purity isabout 92%, about 94%, about 96%, about 98% or about 100% homogeneity.The purity of the peptide conjugates is typically determined by one ormore methods known to those of skill in the art, e.g., liquidchromatography-mass spectrometry (LC-MS), matrix assisted laserdesorption mass time of flight spectrometry (MALDITOF), capillaryelectrophoresis, and the like.

“Substantially uniform glycoform” or a “substantially uniformglycosylation pattern,” when referring to a glycopeptide species, refersto the percentage of acceptor moieties that are glycosylated by theglycosyltransferase of interest (e.g., fucosyltransferase). For example,in the case of a α1,2 fucosyltransferase, a substantially uniformfucosylation pattern exists if substantially all (as defined below) ofthe Galβ1,4-GlcNAc-R and sialylated analogues thereof are fucosylated ina peptide conjugate of the invention. It will be understood by one ofskill in the art, that the starting material may contain glycosylatedacceptor moieties (e.g., fucosylated Galβ1,4-GlcNAc-R moieties). Thus,the calculated percent glycosylation will include acceptor moieties thatare glycosylated by the methods of the invention, as well as thoseacceptor moieties already glycosylated in the starting material.

The term “substantially” in the above definitions of “substantiallyuniform” generally means at least about 40%, at least about 70%, atleast about 80%, or more preferably at least about 90%, and still morepreferably at least about 95% of the acceptor moieties for a particularglycosyltransferase are glycosylated.

Introduction

The present invention provides stabilized peptides for therapeutic use.In one embodiment the invention provides conjugates of glycopeptides inwhich a modified sugar moiety is attached either directly or indirectly(e.g., through and intervening glycosyl residue) to an O-linkedglycosylation site on the peptide. Also provided are methods forproducing the conjugates of the invention.

The O-linked glycosylation site is generally the hydroxy side chain of anatural (e.g., serine, threonine) or unnatural (e.g., 5-hydroxyprolineor 5-hydroxylysine) amino acid. Exemplary O-linked saccharyl residuesinclude N-acetylgalactosamine, galactose, mannose, GlcNAc, glucose,fucose or xylose.

The methods of the invention can be practiced on any peptide having anO-linked glycosylation site. For example, in some embodiments themethods are of use to produce O-linked glycoconjugates in which theglycosyl moiety is attached to an O-linked glycosylation site that ispresent in the wild type peptide.

In other embodiments the invention provides novel mutant peptides thatinclude one or more O-linked glycosylation site(s) that is/are notpresent in the wild-type peptide. Also provided are O-linkedglycosylated versions of the mutant peptides, and methods of preparingO-linked glycosylated mutant peptides. Additional methods include theelaboration, trimming back and/or modification of the O-linked glycosylresidue and glycosyl residues that are N-, rather than O-linked.

In an exemplary aspect, the invention provides a mutant peptide havingthe formula:

in which AA is an amino acid with a side chain that includes a hydroxylmoiety. Exemplary hydroxyamino acids are threonine and serine. TheGalNAc moiety is linked to AA through the oxygen atom of the hydroxylmoiety. AA may be present in the wild type peptide or, alternatively, itis added or relocated by mutating the sequence of the wild type peptide.X is a modifying group or it is a saccharyl moiety, e.g., sialyl,galactosyl and Gal-Sia groups. In an exemplary embodiment, in which X isa saccharyl moiety, it includes a modifying group, as discussed herein.

As shown in the formulae above, the glycosylated amino acid can be atthe N- or C-peptide terminus or internal to the peptide sequence.

In another exemplary embodiment, the invention provides a peptideconjugate having the formula:

in which Z is a bond or a saccharyl residue selected from Gal, Sia andGal-Sia. Y is a modifying group. The saccharyl residue bearing themodifying group (“glycosyl linking group”) is enzymatically attached tothe peptide-tethered glycosyl residue, e.g., forming an intact glycosyllinking group between the modified sugar and the remainder of thepeptide-tethered glycosyl residue.

In yet another exemplary embodiment, AA is located within a proline-richsegment of the mutant peptide and/or it is proximate a proline residue.Appropriate sequences forming O-linked glycosylation sites are readilydetermined by interrogating the enzymatic O-linked glycosylation ofshort peptides containing one or more putative O-linked glycosylationsites. In another exemplary embodiment, O-linked glycosylation sites canbe created at any position in a molecule, using techniques well known inthe art. Peptides with introduced O-linked glycosylation sites can betested for biological activity according to the methods of theinvention.

The conjugates of the invention are formed between peptides and diversespecies such as water-soluble polymers, therapeutic moieties, diagnosticmoieties, targeting moieties and the like. Also provided are conjugatesthat include two or more peptides linked together through a linker arm,i.e., multifunctional conjugates; at least one peptide beingO-glycosylated or including a mutant O-linked glycosylation site. Themulti-functional conjugates of the invention can include two or morecopies of the same peptide or a collection of diverse peptides withdifferent structures, and/or properties. In exemplary conjugatesaccording to this embodiment, the linker between the two peptides isattached to at least one of the peptides through an O-linked glycosylresidue, such as an O-linked glycosyl intact glycosyl linking group.

The conjugates of the invention are formed by the enzymatic attachmentof a modified sugar to the glycosylated or unglycosylated peptide. Themodified sugar is directly added to an O-linked glycosylation site, orto a glycosyl residue attached either directly or indirectly (e.g.,through one or more glycosyl residue) to an O-linked glycosylation site.The invention also provides a conjugate of an O-linked glycosylatedpeptide in which a modified sugar is directly attached to an N-linkedsite, or to a glycosyl residue attached either directly or indirectly toan N-linked glycosylation site.

The modified sugar, when interposed between the peptide (or glycosylresidue) and the modifying group on the sugar becomes what is referredto herein as “an intact glycosyl linking group.” Using the exquisiteselectivity of enzymes, such as glycosyltransferases, the present methodprovides peptides that bear a desired group at one or more specificlocations. Thus, according to the present invention, a modified sugar isattached directly to a selected locus on the peptide chain or,alternatively, the modified sugar is appended onto a carbohydrate moietyof a glycopeptide. Peptides in which modified sugars are bound to both aglycopeptide carbohydrate and directly to an amino acid residue of thepeptide backbone are also within the scope of the present invention.

In contrast to known chemical and enzymatic peptide elaborationstrategies, the methods of the invention, make it possible to assemblepeptides and glycopeptides that have a substantially homogeneousderivatization pattern; the enzymes used in the invention are generallyselective for a particular amino acid residue or combination of aminoacid residues of the peptide. The methods are also practical forlarge-scale production of modified peptides and glycopeptides. Thus, themethods of the invention provide a practical means for large-scalepreparation of glycopeptides having preselected uniform derivatizationpatterns. The methods are particularly well suited for modification oftherapeutic peptidesmay be used to modify glycopeptides that areincompletely glycosylated during production in cell culture cells (e.g.,mammalian cells, insect cells, plant cells, fungal cells, yeast cells,or prokaryotic cells) or transgenic plants or animals. In otherembodiments, the invention may be used to glycosylate peptides, such asGLP-1, that are not glycosylated in the wild type state. In stillfurther embodiments, glycosylation sites can be introduced by mutationat any position along the peptide backbone. The invention furtherprovides method for testing the biological activity of mutants withintroduced glycosylation sites.

The methods of the invention also provide conjugates of glycosylated andunglycosylated peptides with increased therapeutic half-life due to, forexample, reduced clearance rate, or reduced rate of uptake by the immuneor reticuloendothelial system (RES). Moreover, the methods of theinvention provide a means for masking antigenic determinants onpeptides, thus reducing or eliminating a host immune response againstthe peptide. Selective attachment of targeting agents to a peptide usingan appropriate modified sugar can also be used to target a peptide to aparticular tissue or cell surface receptor that is specific for theparticular targeting agent. Moreover, there is provided a class ofpeptides that are specifically modified with a therapeutic moietyconjugated through a glycosyl linking group.

O-Glycosylation

The present invention provides O-linked glycosylated peptides,conjugates of these species and methods for forming O-linkedglycosylated peptides that include a selected amino acid sequence (“anO-linked glycosylation site”). Of particular interest are mutantpeptides that include an O-linked glycosylation site that is not presentin the wild type peptide. The O-linked glycosylation site is a locus forattachment of a glycosyl residue that bears a modifying group.

Mucin-type O-linked glycosylation, one of the most abundant forms ofprotein glycosylation, is found on secreted and cell surface associatedglycoproteins of all eukaryotic cells. There is great diversity in thestructures created by O-linked glycosylation (hundreds of potentialstructures), which are produced by the catalytic activity of hundreds ofglycosyltransferase enzymes that are resident in the Golgi complex.Diversity exists at the level of the glycan structure and in positionsof attachment of O-glycans to protein backbones. Despite the high degreeof potential diversity, it is clear that O-linked glycosylation is ahighly regulated process that shows a high degree of conservation amongmulticellular organisms.

The first step in mucin-type O-linked glycosylation is catalysed by oneor more members of a large family of UDP-GalNAc: polypeptideN-acetylgalactosaminyltransferases (GalNAc-transferases) (EC 2.4.1.41),which transfer GalNAc to serine and threonine acceptor sites (Hassan etal., J. Biol. Chem. 275: 38197-38205 (2000)). To date twelve members ofthe mammalian GalNAc-transferase family have been identified andcharacterized (Schwientek et al., J. Biol. Chem. 277: 22623-22638(2002)), and several additional putative members of this gene familyhave been predicted from analysis of genome databases. TheGalNAc-transferase isoforms have different kinetic properties and showdifferential expression patterns temporally and spatially, suggestingthat they have distinct biological functions (Hassan et al., J. Biol.Chem. 275: 38197-38205 (2000)). Sequence analysis of GalNAc-transferaseshave led to the hypothesis that these enzymes contain two distinctsubunits: a central catalytic unit, and a C-terminal unit with sequencesimilarity to the plant lectin ricin, designated the “lectin domain”(Hagen et al., J. Biol. Chem. 274: 6797-6803 (1999); Hazes, Protein Eng.10: 1353-1356 (1997); Breton et al., Curr. Opin. Struct. Biol. 9:563-571 (1999)). Previous experiments involving site-specificmutagenesis of selected conserved residues confirmed that mutations inthe catalytic domain eliminated catalytic activity. In contrast,mutations in the “lectin domain” had no significant effects on catalyticactivity of the GalNAc-transferase isoform, GalNAc-T1 (Tenno et al., J.Biol. Chem. 277(49): 47088-96 (2002)). Thus, the C-terminal “lectindomain” was believed not to be functional and not to play roles for theenzymatic functions of GalNAc-transferases (Hagen et al., J. Biol. Chem.274: 6797-6803 (1999)).

However, recent evidence demonstrates that some GalNAc-transferasesexhibit unique activities with partially GalNAc-glycosylatedglycopeptides. The catalytic actions of at least threeGalNAc-transferase isoforms, GalNAc-T4, -T7, and -T10, selectively acton glycopeptides corresponding to mucin tandem repeat domains where onlysome of the clustered potential glycosylation sites have been GalNAcglycosylated by other GalNAc-transferases (Bennett et al., FEBS Letters460: 226-230 (1999); Ten Hagen et al., J. Biol. Chem. 276: 17395-17404(2001); Bennett et al., J. Biol. Chem. 273: 30472-30481 (1998); TenHagen et al., J. Biol. Chem. 274: 27867-27874 (1999)). GalNAc-T4 and -T7recognize different GalNAc-glycosylated peptides and catalyse transferof GalNAc to acceptor substrate sites in addition to those that werepreviously utilized. One of the functions of such GalNAc-transferaseactivities is predicted to represent a control step of the density ofO-glycan occupancy in mucins and mucin-like glycoproteins with highdensity of O-linked glycosylation.

One example of this is the glycosylation of the cancer-associated mucinMUC1. MUC1 contains a tandem repeat O-linked glycosylated region of 20residues (HGVTSAPDTRPAPGSTAPPA) with five potential O-linkedglycosylation sites. GalNAc-T1, -T2, and -T3 can initiate glycosylationof the MUC1 tandem repeat and incorporate at only three sites(HGVTSAPDTRPAPGSTAPPA, GalNAc attachment sites underlined). GalNAc-T4 isunique in that it is the only GalNAc-transferase isoform identified sofar that can complete the O-linked glycan attachment to all fiveacceptor sites in the 20 amino acid tandem repeat sequence of the breastcancer associated mucin, MUC1. GalNAc-T4 transfers GalNAc to at leasttwo sites not used by other GalNAc-transferase isoforms on theGalNAc₄TAP24 glycopeptide (TAPPAHGVTSAPDTRPAPGSTAPP, unique GalNAc-T4attachment sites are in bold) (Bennett et al., J. Biol. Chem. 273:30472-30481 (1998). An activity such as that exhibited by GalNAc-T4appears to be required for production of the glycoform of MUC1 expressedby cancer cells where all potential sites are glycosylated (Muller etal., J. Biol. Chem. 274: 18165-18172 (1999)). Normal MUC1 from lactatingmammary glands has approximately 2.6 O-linked glycans per repeat (Mulleret al., J. Biol. Chem. 272: 24780-24793 (1997) and MUC1 derived from thecancer cell line T47D has 4.8 O-linked glycans per repeat (Muller etal., J. Biol. Chem. 274: 18165-18172 (1999)). The cancer-associated formof MUC1 is therefore associated with higher density of O-linked glycanoccupancy and this is accomplished by a GalNAc-transferase activityidentical to or similar to that of GalNAc-T4.

Polypeptide GalNAc-transferases, which have not displayed apparentGalNAc-glycopeptide specificities, also appear to be modulated by theirputative lectin domains (PCT WO 01/85215 A2). Recently, it was foundthat mutations in the GalNAc-TI putative lectin domain, similarly tothose previously analysed in GalNAc-T4 (Hassan et al., J. Biol. Chem.275: 38197-38205 (2000)), modified the activity of the enzyme in asimilar fashion as GalNAc-T4. Thus, while wild type GalNAc-TI addedmultiple consecutive GalNAc residues to a peptide substrate withmultiple acceptor sites, mutated GalNAc-T1 failed to add more than oneGalNAc residue to the same substrate (Tenno et al., J. Biol. Chem.277(49): 47088-96 (2002)).

Since it has been demonstrated that mutations of GalNAc transferases canbe utilized to produce glycosylation patterns that are distinct fromthose produced by the wild-type enzymes, it is within the scope of thepresent invention to utilize one or more mutant GalNAc transferase inpreparing the O-linked glycosylated peptides of the invention.

Mutant GLP-1 Peptides with O-linked Glycosylation Sites

The peptides provided by the present invention include an amino acidsequence that is recognized as a GalNAc acceptor by one or morewild-type or mutant GalNAc transferases. The amino acid sequence of thepeptide is either the wild-type, for those peptides that include anO-linked glycosylation site, or may be a mutant sequence in which anon-naturally occurring O-linked glycosylation site is introduced. Anexemplary peptide with which the present invention is practiced includesGlucagon-Like Peptide-1 (GLP-1). The emphasis of the followingdiscussion on GLP-1 is for clarity of illustration. Those of skill willunderstand that the strategy set forth herein for preparing O-linkedglycoconjugated analogues of wild-type and mutant peptides is applicableto any peptide.

In an exemplary embodiment, the peptide is a biologically active GLP-1mutant that includes one or more mutations at one or more sitesdistributed along the peptide backbone. Representative wild type andmutant GLP-1 polypeptides of the invention have sequences that areselected from:

GLP-1 Glycopeptides

Ac-X-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂H-X-EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂HA-X-GTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂HAE-X-TFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂HAEG-X-FTSDVSSYLEGQAAKEFIAWLVKGR-NH₂HAEGT-X-TSDVSSYLEGQAAKEFIAWLVKGR-NH₂HAEGTF-X-SDVSSYLEGQAAKEFIAWLVKGR-NH₂HAEGTFT-X-DVSSYLEGQAAKEFIAWLVKGR-NH₂HAEGTFTS-X-VSSYLEGQAAKEFIAWLVKGR-NH₂HAEGTFTSD-X-SSYLEGQAAKEFIAWLVKGR-NH₂HAEGTFVSDV-X-SYLEGQAAKEFIAWLVKGR-NH₂HAEGTFTSDVS-X-YLEGQAAKEFIAWLVKGR-NH₂HAEGTFTSDVSS-X-LEGQAAKEFIAWLVKGR-NH₂HAEGTFTSDVSSY-X-EGQAAKEFIAWLVKGR-NH₂HAEGTFTSDVSSYL-X-GQAAKEFIAWLVKGR-NH₂HAEGTFTSDVSSYLE-X-QAAKEFIAWLVKGR-NH₂HAEGTFTSDVSSYLEG-X-AAKEFIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQ-X-AKEFIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQA-X-KEFIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQAA-X-EFIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQAAK-X-FIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQAAKE-X-IAWLVKGR-NH₂HAEGTFTSDVSSYLEGQAAKEF-X-AWLVKGR-NH₂HAEGTFTSDVSSYLEGQAAKEFI-X-WLVKGR-NH₂HAEGTFTSDVSSYLEGQAAKEFIA-X-LVKGR-NH₂HAEGTFTSDVSSYLEGQAAKEFIAW-X-VKGR-NH₂HAEGTFTSDVSSYLEGQAAKEFIAWL-X-KGR-NH₂HAEGTFTSDVSSYLEGQAAKEFIAWLV-X-GR-NH₂HAEGTFTSDVSSYLEGQAAKEFIAWLVK-X-R-NH₂ HAEGTFTSDVSSYLEGQAAKEFIAWLVKG-X-NH₂HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-X-NH₂

In another exemplary embodiment, the peptide is a fusion of one or morepeptides. In another exemplary embodiment, the components of the peptideare members selected from a GLP-1, GLP-1 analogs and/or GLP-1 mutants.In another exemplary embodiment, the components of the peptide are oneor more non-GLP-1 peptides and GLP-1, GLP-1 analog and/or GLP-1 mutant.In another exemplary embodiment, the peptide is an Oxyntomodulin-GLP-1fusion. This peptide has the following sequence.

HSQGTFTSDY SKYLDSRRAQ DFVQWLMNTK RNRNNIAKRH DEFERHAEGT FTSDVSSYLEGQAAKEFIAW LVKGRG

In another exemplary embodiment, the peptide is an oxyntomodulin/GLP-1mutant fusion. In another exemplary embodiment, oxyntomodulin/GLP-1mutant fusions have the following “natural” sequence

-----T²⁹KRNRNNIAKRHDEFERHAE-----, natural sequence;replaced with sequences that are selected from:

T²⁹ BJJ′RN(Z′)_(a)NIAOUXX′O′FEZHAEwherein all substitutions are independently selected from:

-   -   B=N (natural human variant), K, A, G, S, T, L    -   J=R, G, A, S, T, L    -   O=K, P    -   U=T, S, K    -   X=H, A, Q, N, G, or any uncharged amino acid    -   X′=D, G, A, N, E, or any uncharged amino acid    -   Z=R, A, G, S, T, V, I, L or any uncharged amino acid    -   Z′=G, A    -   J′=N, S, T    -   O′=E, A, G, M, any uncharged amino acid    -   a=0 or 1        Representative examples of oxyntomodulin/GLP-1 mutant fusions        have the following natural sequence

-----T²⁹KRNRNNIAKRHDEFERHAE-----, natural sequence;replaced with sequences that are selected from:

--T²⁹NANRNNIAPTHDEFEAHAE-- --T²⁹NANRNNIAPTQDEFEAHAE----T²⁹NANRNNIAPTTDEFEAHAE-- --T²⁹NANRNNIAPTQGEFEAHAE----T²⁹NANRNNIAPTQGAFEAHAE-- --T²⁹NANRNNIAPTQGAMPAHAE----T²⁹ARNRNNIAPTQGAMEAHAE-- --T²⁹NANRNNLAPTINTFEAHAE----T²⁹NANRNNIAPTQAYSEGHAE-- --T²⁹NANRNNIAPTQAYFEGHAE----T²⁹NANRNNIAPTTASFEGHAE-- --T²⁹NANRNNIAPTTLYVEGHAE----T²⁹ARNRNNIAPTINTFEGHAE-- --T²⁹NANRNNIAPTINTFEGHAE----T²⁹NANRSGDIPTINTFEGHAE--These sequences are based on human sequences in an attempt to minimizeimmunogenicity while creating a site for glycosylation and preventingproteolysis.

In another exemplary embodiment, one of the non-GLP-1 peptides is amember selected from a GLP-2, GLP-2 analog and/or GLP-2 mutant. Inanother exemplary embodiment, the peptide is a GLP-1/GLP-2 fusion. Thispeptide has the following sequence.

HAE GTFTSDVSSY LEGQAAKEFI AWLVKGRGRR DFPEEVAIVE ELGRRHADGS FSDEMNTILDNLAARDFINW LIQTKITDRKIn another exemplary embodiment, the peptide is a GLP-1/GLP-2 mutantfusion. In another exemplary embodiment, GLP-1/GLP-2 mutant fusions havethe following “natural” sequence

HA--R³⁰GRRDFPEEVAIVEELGRRHADG--, natural sequence;replaced with sequences that are selected from:

HX″--R³⁰GBB′DFPOU(O′)_(a) JJ′VEELGZZ′HADG-wherein all substitutions are independently selected from:

-   -   B and B′ (independently selected)=R, A, G, V, I, L, Q, P    -   J=P, A, 1, V, G    -   O=T, S, E    -   U=E, S, T, Q, 1, V, L, and uncharged amino acid    -   X″=A, G, S, T    -   Z and Z′ (independently selected)=R, A, G, S, T, V, I, L or any        uncharged amino acid    -   J′=E, Y, 1, N, A, F, G, or any uncharged amino acid    -   O′=SLP, NT, Y, V, Y    -   a=0 or 1        Representative examples of GLP-1/GLP-2 mutant fusions have the        following natural sequence    -   HA—R³⁰ GRRDFPEEVAIVEELGRRHADG—, natural sequence; replaced with        sequences that are selected from:

HS--R³⁰GQPDFPEG S LPVAIVEELGRGHADG- HS--R³⁰GQPDFP T GSLPVAIVEELGRGHADG-HS--R³⁰GQPDFP T TSEPVAIVEELGRGHADG- HS--R³⁰GQPDFP T AVIPVAIVEELGRGHADG-HS--R³⁰GQPDFPG ST APVAIVEELGRGHADG- HS--R³⁰GQPDFPL T LEPVAIVEELGRGHADG-HS--R³⁰GQPDFP T SGEPVAIVEELGRGHADG- HS--R³⁰GQPDFP T INTPVAIVEELGRGHADG-HS--R³⁰GQPDFP T TLYPVAIVEELGRGHADG- HS--R³⁰GQPDFPEGSLP T AIVEELGRGHADG-HS--R³⁰GQPDFPEGSLP T INTEELGRGHADG- HS--R³⁰GQPDFPEGSLP T QAVEELGRGHADG--HS--R³⁰GQADFPEEVP T VEELGRGHADG- HS--R³⁰GQADFPEEVP T INTLGRGHADG-HS--R³⁰GQADFPEEVP T QGALGRGHADG- HS--R³⁰GQADFPEEVP T TLYLGRGHADG-HS--R³⁰GQADFP T VLPIVEELGRGHADG- HS--R³⁰GQADFP T EIPIVEELGRGHADG-HS--R³⁰GQADFP S DGPIVEELGRGHADG- HS--R³⁰GQADFP T EVPIVEELGRGHADG--These sequences are based on human sequences in an attempt to minimizeimmunogenicity while creating a site for glycosylation and preventingproteolysis.

In another exemplary embodiment, the peptide is a fusion of threepeptides, thus forming a triple fusion. The three peptides can bearranged in any order. In another exemplary embodiment, the threepeptides are oxyntomodulin, GLP-1 and GLP-2. This peptide has thefollowing sequence.

HSQGTFTS DYSKYLDSRR AQDFVQWLMN TKRNRNNIKRHDEFERHAE GTFTSDVSSY LEGQAAKEFI AWLVKGRGRR DFPEEVAIVEELGRRHADGS FSDEMNTILD NLAARDFINW LIQTKITDRKIn another exemplary embodiment, the peptide is anoxyntomodulin/GLP-1/GLP-2 mutant fusion. In another exemplaryembodiment, an oxyntomodulin/GLP-1/GLP-2 mutant fusion has the following“natural” sequence

HS-----T²⁹KRNRNNIAKRHDEFERHAE---H³⁶A-- R⁶⁵GRRDFPEEVAIVEELGRRHADG--,natural sequence;replaced with sequences that are selected from:

HS--T²⁹ BJJ′RN(Z′)_(a)NIAOUXX′O′FEZHAE-- R⁶⁶GB′B″DFPO″U′(O″′)_(a)J″J″′VEELGX″′Z″HADG--wherein all substitutions are independently selected from:

-   -   B=N (natural human variant), K, A, G, S, T, L    -   J=R, G, A, S, T, L    -   O=K, P    -   U=T, S    -   X=H, A, Q, N, G, or any uncharged amino acid    -   X′=D, G, A, N, E, or any uncharged amino acid    -   Z=R, A, G, S, T, V, I, L or any uncharged amino acid    -   Z′=G, A    -   J′=N, S, T    -   O′=E, A, G, M, any uncharged amino acid    -   a=0 or 1    -   B″ and B′ (independently selected)=R, A, G, V, I, L, Q, P    -   J″=P, A, I, V, G    -   O″=T, S, E    -   U′=E, S, T, Q, I, V, L, and uncharged amino acid    -   X″=A, G, S, T    -   X′″ and Z″ (independently selected)=R, A, G, S, T, V, I, L or        any uncharged amino acid    -   J′″=E, Y, I, N, A, F, G, or any uncharged amino acid    -   O′″=SLP, NT, Y, V, Y        Representative examples of an oxyntomodulin/GLP-1/GLP-2 mutant        fusion have the following “natural” sequence

HS-----T²⁹KRNRNNIAKRHDEFERHAE---H³⁶A-- R⁶⁵GRRDFPEEVAIVEELGRRHADG--,natural sequence;replaced with sequences that are selected from:

HS--T²⁹NANRS G DIPKAHDEFEAHAE-- R⁶⁶GQPDFPEG S LPVAIVEELGRGHADG-HS--T²⁹NANRSDIPKAHDEFEAHAE-- R⁶⁶GQPDFPEG S LPVAIVEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQPDFP T GSLPVAIVEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQPDFP T TSEPVAIVEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQPDFP T AVIPVAIVEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQPDFPG ST APVAIVEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQPDFPL T LEPVAIVEELGRGHADG-HS--T²⁹NANANINIAKAHDEFEAHAE-- R⁶⁶GQPDFP T SGEPVAIVEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQPDFP T INTPVAIVEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQPDFP T TLYPVAIVEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQPDFPEGSLP T AIVEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQPDFPEGSLP T INTEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQPDFPEGSLP T QAVEELGRGHADG--HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQADFPEEVP T VEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQADFPEEVP T INTLGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQADFPEEVP T QGALGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQADFPEEVP T TLYLGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQADFP T VLPIVEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQADFP T EIPIVEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQADFP S DGPIVEELGRGHADG-HS--T²⁹NANANNIAKAHDEFEAHAE-- R⁶⁶GQADFP T EVPIVEELGRGHADG--These sequences are based on human sequences in an attempt to minimizeimmunogenicity while creating a site for glycosylation and preventingproteolysis.

Appropriate O-linked glycosylation sequences for GLP-1 and peptidesother than GLP-1 can be determined by preparing a polypeptideincorporating a putative O-linked glycosylation site and submitting thatpolypeptide to suitable O-linked glycosylation conditions, therebyconfirming its ability to serve as an acceptor for a GalNAc transferase.Moreover, as will be apparent to one of skill in the art, peptides thatinclude one or more mutations are within the scope of the presentinvention. The mutations are designed to allow the adjustment ofdesirable properties of the peptides, e.g., activity and number andposition of O- and/or N-linked glycosylation sites on the peptide.

Acquisition of Peptide Coding Sequences General Recombinant Technology

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook and Russell, Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression. ALaboratory Manual (1990); and Ausubel et al., eds., Current Protocols inMolecular Biology (1994).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized, e.g., according to the solid phase phosphoramidite triestermethod first described by Beaucage & Caruthers, Tetrahedron Lett. 22:1859-1862 (1981), using an automated synthesizer, as described in VanDevanter et. al., Nucleic Acids Res. 12: 6159-6168 (1984). Entire genescan also be chemically synthesized. Purification of oligonucleotides isperformed using any art-recognized strategy, e.g., native acrylamide gelelectrophoresis or anion-exchange HPLC as described in Pearson &Reanier, J Chrom. 255: 137-149 (1983).

The sequence of the cloned wild-type peptide genes, polynucleotideencoding mutant peptides, and synthetic oligonucleotides can be verifiedafter cloning using, e.g., the chain termination method for sequencingdouble-stranded templates of Wallace et al., Gene 16: 21-26 (1981).

Cloning and Subcloning of a Wild-Type Peptide Coding Sequence

Numerous polynucleotide sequences encoding wild-type peptides have beendetermined and are available from a commercial supplier, e.g., humangrowth hormone, e.g., GenBank Accession Nos. NM 000515, NM 002059, NM022556, NM 022557, NM 022558, NM 022559, NM 022560, NM 022561, and NM022562.

The rapid progress in the studies of human genome has made possible acloning approach where a human DNA sequence database can be searched forany gene segment that has a certain percentage of sequence homology to aknown nucleotide sequence, such as one encoding a previously identifiedpeptide. Any DNA sequence so identified can be subsequently obtained bychemical synthesis and/or a polymerase chain reaction (PCR) techniquesuch as overlap extension method. For a short sequence, completely denovo synthesis may be sufficient; whereas further isolation of fulllength coding sequence from a human cDNA or genomic library using asynthetic probe may be necessary to obtain a larger gene.

Alternatively, a nucleic acid sequence encoding a peptide can beisolated from a human cDNA or genomic DNA library using standard cloningtechniques such as polymerase chain reaction (PCR), where homology-basedprimers can often be derived from a known nucleic acid sequence encodinga peptide. Most commonly used techniques for this purpose are describedin standard texts, e.g., Sambrook and Russell, supra.

cDNA libraries suitable for obtaining a coding sequence for a wild-typepeptide may be commercially available or can be constructed. The generalmethods of isolating mRNA, making cDNA by reverse transcription,ligating cDNA into a recombinant vector, transfecting into a recombinanthost for propagation, screening, and cloning are well known (see, e.g.,Gubler and Hoffman, Gene, 25: 263-269 (1983); Ausubel et al., supra).Upon obtaining an amplified segment of nucleotide sequence by PCR, thesegment can be further used as a probe to isolate the full-lengthpolynucleotide sequence encoding the wild-type peptide from the cDNAlibrary. A general description of appropriate procedures can be found inSambrook and Russell, supra.

A similar procedure can be followed to obtain a full length sequenceencoding a wild-type peptide, e.g., any one of the GenBank Accession Nosmentioned above, from a human genomic library. Human genomic librariesare commercially available or can be constructed according to variousart-recognized methods. In general, to construct a genomic library, theDNA is first extracted from an tissue where a peptide is likely found.The DNA is then either mechanically sheared or enzymatically digested toyield fragments of about 12-20 kb in length. The fragments aresubsequently separated by gradient centrifugation from polynucleotidefragments of undesired sizes and are inserted in bacteriophage λvectors. These vectors and phages are packaged in vitro. Recombinantphages are analyzed by plaque hybridization as described in Benton andDavis, Science, 196: 180-182 (1977). Colony hybridization is carried outas described by Grunstein et al., Proc. Natl. Acad. Sci. USA, 72:3961-3965 (1975).

Based on sequence homology, degenerate oligonucleotides can be designedas primer sets and PCR can be performed under suitable conditions (see,e.g., White et al., PCR Protocols. Current Methods and Applications,1993; Griffin and Griffin, PCR Technology, CRC Press Inc. 1994) toamplify a segment of nucleotide sequence from a cDNA or genomic library.Using the amplified segment as a probe, the full-length nucleic acidencoding a wild-type peptide is obtained.

Upon acquiring a nucleic acid sequence encoding a wild-type peptide, thecoding sequence can be subcloned into a vector, for instance, anexpression vector, so that a recombinant wild-type peptide can beproduced from the resulting construct. Further modifications to thewild-type peptide coding sequence, e.g., nucleotide substitutions, maybe subsequently made to alter the characteristics of the molecule.

Introducing Mutations into a Peptide Sequence

From an encoding polynucleotide sequence, the amino acid sequence of awild-type peptide can be determined. Subsequently, this amino acidsequence may be modified to alter the protein's glycosylation pattern,by introducing additional glycosylation site(s) at various locations inthe amino acid sequence.

Several types of protein glycosylation sites are well known in the art.For instance, in eukaryotes, N-linked glycosylation occurs on theasparagine of the consensus sequence Asn-X_(aa)-Ser/Thr, in which X_(aa)is any amino acid except proline (Kornfeld et al., Ann Rev Biochem54:631-664 (1985); Kukuruzinska et al., Proc. Natl. Acad. Sci. USA84:2145-2149 (1987); Herscovics et al., FASEB J 7:540-550 (1993); andOrlean, Saccharomyces Vol. 3 (1996)). O-linked glycosylation takes placeat serine or threonine residues (Tanner et al., Biochim. Biophys. Acta.906:81-91 (1987); and Hounsell et al., Glycoconj. J 13:19-26 (1996)).Other glycosylation patterns are formed by linkingglycosylphosphatidylinositol to the carboxyl-terminal carboxyl group ofthe protein (Takeda et al., Trends Biochem. Sci. 20:367-371 (1995); andUdenfriend et al., Ann. Rev. Biochem. 64:593-591 (1995). Based on thisknowledge, suitable mutations can thus be introduced into a wild-typepeptide sequence to form new glycosylation sites.

Although direct modification of an amino acid residue within a peptidepolypeptide sequence may be suitable to introduce a new N-linked orO-linked glycosylation site, more frequently, introduction of a newglycosylation site is accomplished by mutating the polynucleotidesequence encoding a peptide. This can be achieved by using any of knownmutagenesis methods, some of which are discussed below. Exemplarymodifications to a GLP-1 peptide include those illustrated in SEQ ID NO:______.

A variety of mutation-generating protocols are established and describedin the art. See, e.g., Zhang et al., Proc. Natl. Acad. Sci. USA, 94:4504-4509 (1997); and Stemmer, Nature, 370: 389-391 (1994). Theprocedures can be used separately or in combination to produce variantsof a set of nucleic acids, and hence variants of encoded polypeptides.Kits for mutagenesis, library construction, and otherdiversity-generating methods are commercially available.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Botstein and Shortle, Science, 229: 1193-1201(1985)), mutagenesis using uracil-containing templates (Kunkel, Proc.Natl. Acad. Sci. USA, 82: 488-492 (1985)), oligonucleotide-directedmutagenesis (Zoller and Smith, Nucl. Acids Res., 10: 6487-6500 (1982)),phosphorothioate-modified DNA mutagenesis (Taylor et al., Nucl. AcidsRes., 13: 8749-8764 and 8765-8787 (1985)), and mutagenesis using gappedduplex DNA (Kramer et al., Nucl. Acids Res., 12: 9441-9456 (1984)).

Other methods for generating mutations include point mismatch repair(Kramer et al., Cell, 38: 879-887 (1984)), mutagenesis usingrepair-deficient host strains (Carter et al., Nucl. Acids Res., 13:4431-4443 (1985)), deletion mutagenesis (Eghtedarzadeh and Henikoff,Nuc. Acids Res., 14: 5115 (1986)), restriction-selection andrestriction-purification (Wells et al., Phil. Trans. R. Soc. Lond. A,317: 415-423 (1986)), mutagenesis by total gene synthesis (Nambiar etal., Science, 223: 1299-1301 (1984)), double-strand break repair(Mandecki, Proc. Natl. Acad. Sci. USA, 83: 7177-7181 (1986)),mutagenesis by polynucleotide chain termination methods (U.S. Pat. No.5,965,408), and error-prone PCR (Leung et al., Biotechniques, 1: 11-15(1989)).

Modification of Nucleic Acids for Preferred Codon Usage in a HostOrganism

The polynucleotide sequence encoding a mutant peptide can be furtheraltered to coincide with the preferred codon usage of a particular host.For example, the preferred codon usage of one strain of bacterial cellscan be used to derive a polynucleotide that encodes a mutant peptide ofthe invention and includes the codons favored by this strain. Thefrequency of preferred codon usage exhibited by a host cell can becalculated by averaging frequency of preferred codon usage in a largenumber of genes expressed by the host cell (e.g., calculation service isavailable from web site of the Kazusa DNA Research Institute, Japan).This analysis is preferably limited to genes that are highly expressedby the host cell. U.S. Pat. No. 5,824,864, for example, provides thefrequency of codon usage by highly expressed genes exhibited bydicotyledonous plants and monocotyledonous plants.

At the completion of modification, the mutant peptide coding sequencesare verified by sequencing and are then subcloned into an appropriateexpression vector for recombinant production in the same manner as thewild-type peptides.

Expression and Purification of the Mutant Peptide

Following sequence verification, the mutant peptide of the presentinvention can be produced using routine techniques in the field ofrecombinant genetics, relying on the polynucleotide sequences encodingthe polypeptide disclosed herein.

Expression Systems

To obtain high-level expression of a nucleic acid encoding a mutantpeptide of the present invention, one typically subclones apolynucleotide encoding the mutant peptide into an expression vectorthat contains a strong promoter to direct transcription, atranscription/translation terminator and a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook and Russell, supra, and Ausubelet al., supra. Bacterial expression systems for expressing the wild-typeor mutant peptide are available in, e.g., E. coli, Bacillus sp.,Salmonella, and Caulobacter. Kits for such expression systems arecommercially available. Eukaryotic expression systems for mammaliancells, yeast, and insect cells are well known in the art and are alsocommercially available. In one embodiment, the eukaryotic expressionvector is an adenoviral vector, an adeno-associated vector, or aretroviral vector.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is optionallypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically includes atranscription unit or expression cassette that contains all theadditional elements required for the expression of the mutant peptide inhost cells. A typical expression cassette thus contains a promoteroperably linked to the nucleic acid sequence encoding the mutant peptideand signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. The nucleic acidsequence encoding the peptide is typically linked to a cleavable signalpeptide sequence to promote secretion of the peptide by the transformedcell. Such signal peptides include, among others, the signal peptidesfrom tissue plasminogen activator, insulin, and neuron growth factor,and juvenile hormone esterase of Heliothis virescens. Additionalelements of the cassette may include enhancers and, if genomic DNA isused as the structural gene, introns with functional splice donor andacceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322-based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 later promoter, metallothionein promoter, murine mammary tumorvirus promoter, Rous sarcoma virus promoter, polyhedrin promoter, orother promoters shown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as abaculovirus vector in insect cells, with a polynucleotide sequenceencoding the mutant peptide under the direction of the polyhedrinpromoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are optionally chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

When periplasmic expression of a recombinant protein (e.g., a hgh mutantof the present invention) is desired, the expression vector furthercomprises a sequence encoding a secretion signal, such as the E. coliOppA (Periplasmic Oligopeptide Binding Protein) secretion signal or amodified version thereof, which is directly connected to 5′ of thecoding sequence of the protein to be expressed. This signal sequencedirects the recombinant protein produced in cytoplasm through the cellmembrane into the periplasmic space. The expression vector may furthercomprise a coding sequence for signal peptidase 1, which is capable ofenzymatically cleaving the signal sequence when the recombinant proteinis entering the periplasmic space. More detailed description forperiplasmic production of a recombinant protein can be found in, e.g.,Gray et al., Gene 39: 247-254 (1985), U.S. Pat. Nos. 6,160,089 and6,436,674.

As discussed above, a person skilled in the art will recognize thatvarious conservative substitutions can be made to any wild-type ormutant peptide or its coding sequence while still retaining thebiological activity of the peptide. Moreover, modifications of apolynucleotide coding sequence may also be made to accommodate preferredcodon usage in a particular expression host without altering theresulting amino acid sequence.

Transfection Methods

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of the mutantpeptide, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264: 17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA, or other foreign genetic material into a host cell (see,e.g., Sambrook and Russell, supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingthe mutant peptide.

Detection of Expression of Mutant Peptide in Host Cells

After the expression vector is introduced into appropriate host cells,the transfected cells are cultured under conditions favoring expressionof the mutant peptide. The cells are then screened for the expression ofthe recombinant polypeptide, which is subsequently recovered from theculture using standard techniques (see, e.g., Scopes, ProteinPurification: Principles and Practice (1982); U.S. Pat. No. 4,673,641;Ausubel et al., supra; and Sambrook and Russell, supra).

Several general methods for screening gene expression are well knownamong those skilled in the art. First, gene expression can be detectedat the nucleic acid level. A variety of methods of specific DNA and RNAmeasurement using nucleic acid hybridization techniques are commonlyused (e.g., Sambrook and Russell, supra). Some methods involve anelectrophoretic separation (e.g., Southern blot for detecting DNA andNorthern blot for detecting RNA), but detection of DNA or RNA can becarried out without electrophoresis as well (such as by dot blot). Thepresence of nucleic acid encoding a mutant peptide in transfected cellscan also be detected by PCR or RT-PCR using sequence-specific primers.

Second, gene expression can be detected at the polypeptide level.Various immunological assays are routinely used by those skilled in theart to measure the level of a gene product, particularly usingpolyclonal or monoclonal antibodies that react specifically with amutant peptide of the present invention, such as a polypeptide havingthe amino acid sequence of SEQ ID NO: 1-7, (e.g., Harlow and Lane,Antibodies, A Laboratory Manual, Chapter 14, Cold Spring Harbor, 1988;Kohler and Milstein, Nature, 256: 495-497 (1975)). Such techniquesrequire antibody preparation by selecting antibodies with highspecificity against the mutant peptide or an antigenic portion thereof.The methods of raising polyclonal and monoclonal antibodies are wellestablished and their descriptions can be found in the literature, see,e.g., Harlow and Lane, supra; Kohler and Milstein, Eur. J. Immunol., 6:511-519 (1976). More detailed descriptions of preparing antibody againstthe mutant peptide of the present invention and conducting immunologicalassays detecting the mutant peptide are provided in a later section.

Purification of Recombinantly Produced Mutant Peptide

Once the expression of a recombinant mutant peptide in transfected hostcells is confirmed, the host cells are then cultured in an appropriatescale for the purpose of purifying the recombinant polypeptide.

1. Purification of Recombinantly Produced Mutant Peptide from Bacteria

When the mutant peptides of the present invention are producedrecombinantly by transformed bacteria in large amounts, typically afterpromoter induction, although expression can be constitutive, theproteins may form insoluble aggregates. There are several protocols thatare suitable for purification of protein inclusion bodies. For example,purification of aggregate proteins (hereinafter referred to as inclusionbodies) typically involves the extraction, separation and/orpurification of inclusion bodies by disruption of bacterial cells, e.g.,by incubation in a buffer of about 100-150 μg/ml lysozyme and 0.1%Nonidet P40, a non-ionic detergent. The cell suspension can be groundusing a Polytron grinder (Brinkman Instruments, Westbury, N.Y.).Alternatively, the cells can be sonicated on ice. Alternate methods oflysing bacteria are described in Ausubel et al. and Sambrook andRussell, both supra, and will be apparent to those of skill in the art.

The cell suspension is generally centrifuged and the pellet containingthe inclusion bodies resuspended in buffer which does not dissolve butwashes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA,150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may benecessary to repeat the wash step to remove as much cellular debris aspossible. The remaining pellet of inclusion bodies may be resuspended inan appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mMNaCl). Other appropriate buffers will be apparent to those of skill inthe art.

Following the washing step, the inclusion bodies are solubilized by theaddition of a solvent that is both a strong hydrogen acceptor and astrong hydrogen donor (or a combination of solvents each having one ofthese properties). The proteins that formed the inclusion bodies maythen be renatured by dilution or dialysis with a compatible buffer.Suitable solvents include, but are not limited to, urea (from about 4 Mto about 8 M), formamide (at least about 80%, volume/volume basis), andguanidine hydrochloride (from about 4 M to about 8 M). Some solventsthat are capable of solubilizing aggregate-forming proteins, such as SDS(sodium dodecyl sulfate) and 70% formic acid, may be inappropriate foruse in this procedure due to the possibility of irreversibledenaturation of the proteins, accompanied by a lack of immunogenicityand/or activity. Although guanidine hydrochloride and similar agents aredenaturants, this denaturation is not irreversible and renaturation mayoccur upon removal (by dialysis, for example) or dilution of thedenaturant, allowing re-formation of the immunologically and/orbiologically active protein of interest. After solubilization, theprotein can be separated from other bacterial proteins by standardseparation techniques. For further description of purifying recombinantpeptide from bacterial inclusion body, see, e.g., Patra et al., ProteinExpression and Purification 18: 182-190 (2000).

Alternatively, it is possible to purify recombinant polypeptides, e.g.,a mutant peptide, from bacterial periplasm. Where the recombinantprotein is exported into the periplasm of the bacteria, the periplasmicfraction of the bacteria can be isolated by cold osmotic shock inaddition to other methods known to those of skill in the art (see e.g.,Ausubel et al., supra). To isolate recombinant proteins from theperiplasm, the bacterial cells are centrifuged to form a pellet. Thepellet is resuspended in a buffer containing 20% sucrose. To lyse thecells, the bacteria are centrifuged and the pellet is resuspended inice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant proteins present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art.

2. Standard Protein Separation Techniques for Purification

When a recombinant polypeptide, e.g., the mutant peptide of the presentinvention, is expressed in host cells in a soluble form, itspurification can follow the standard protein purification proceduredescribed below.

i. Solubility Fractionation

Often as an initial step, and if the protein mixture is complex, aninitial salt fractionation can separate many of the unwanted host cellproteins (or proteins derived from the cell culture media) from therecombinant protein of interest, e.g., a mutant peptide of the presentinvention. The preferred salt is ammonium sulfate. Ammonium sulfateprecipitates proteins by effectively reducing the amount of water in theprotein mixture. Proteins then precipitate on the basis of theirsolubility. The more hydrophobic a protein is, the more likely it is toprecipitate at lower ammonium sulfate concentrations. A typical protocolis to add saturated ammonium sulfate to a protein solution so that theresultant ammonium sulfate concentration is between 20-30%. This willprecipitate the most hydrophobic proteins. The precipitate is discarded(unless the protein of interest is hydrophobic) and ammonium sulfate isadded to the supernatant to a concentration known to precipitate theprotein of interest. The precipitate is then solubilized in buffer andthe excess salt removed if necessary, through either dialysis ordiafiltration. Other methods that rely on solubility of proteins, suchas cold ethanol precipitation, are well known to those of skill in theart and can be used to fractionate complex protein mixtures.

ii. Size Differential Filtration

Based on a calculated molecular weight, a protein of greater and lessersize can be isolated using ultrafiltration through membranes ofdifferent pore sizes (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of a protein of interest, e.g., a mutant peptide. The retentateof the ultrafiltration is then ultrafiltered against a membrane with amolecular cut off greater than the molecular weight of the protein ofinterest. The recombinant protein will pass through the membrane intothe filtrate. The filtrate can then be chromatographed as describedbelow.

iii. Column Chromatography

The proteins of interest (such as the mutant peptide of the presentinvention) can also be separated from other proteins on the basis oftheir size, net surface charge, hydrophobicity, or affinity for ligands.In addition, antibodies raised against peptide can be conjugated tocolumn matrices and the peptide immunopurified. All of these methods arewell known in the art.

It will be apparent to one of skill that chromatographic techniques canbe performed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech).

Immunoassays for Detection of Mutant Peptide Expression

To confirm the production of a recombinant mutant peptide, immunologicalassays may be useful to detect in a sample the expression of thepolypeptide. Immunological assays are also useful for quantifying theexpression level of the recombinant hormone. Antibodies against a mutantpeptide are necessary for carrying out these immunological assays.

Production of Antibodies against Mutant Peptide

Methods for producing polyclonal and monoclonal antibodies that reactspecifically with an immunogen of interest are known to those of skillin the art (see, e.g., Coligan, Current Protocols in ImmunologyWiley/Greene, N.Y., 1991; Harlow and Lane, Antibodies: A LaboratoryManual Cold Spring Harbor Press, NY, 1989; Stites et al. (eds.) Basicand Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Goding, Monoclonal Antibodies.Principles and Practice (2d ed.) Academic Press, New York, N.Y., 1986;and Kohler and Milstein Nature 256: 495-497, 1975). Such techniquesinclude antibody preparation by selection of antibodies from librariesof recombinant antibodies in phage or similar vectors (see, Huse et al.,Science 246: 1275-1281, 1989; and Ward et al., Nature 341: 544-546,1989).

In order to produce antisera containing antibodies with desiredspecificity, the polypeptide of interest (e.g., a mutant peptide of thepresent invention) or an antigenic fragment thereof can be used toimmunize suitable animals, e.g., mice, rabbits, or primates. A standardadjuvant, such as Freund's adjuvant, can be used in accordance with astandard immunization protocol. Alternatively, a synthetic antigenicpeptide derived from that particular polypeptide can be conjugated to acarrier protein and subsequently used as an immunogen.

The animal's immune response to the immunogen preparation is monitoredby taking test bleeds and determining the titer of reactivity to theantigen of interest. When appropriately high titers of antibody to theantigen are obtained, blood is collected from the animal and antiseraare prepared. Further fractionation of the antisera to enrich antibodiesspecifically reactive to the antigen and purification of the antibodiescan be performed subsequently, see, Harlow and Lane, supra, and thegeneral descriptions of protein purification provided above.

Monoclonal antibodies are obtained using various techniques familiar tothose of skill in the art. Typically, spleen cells from an animalimmunized with a desired antigen are immortalized, commonly by fusionwith a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol.6:511-519, 1976). Alternative methods of immortalization include, e.g.,transformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and the yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host.

Additionally, monoclonal antibodies may also be recombinantly producedupon identification of nucleic acid sequences encoding an antibody withdesired specificity or a binding fragment of such antibody by screeninga human B cell cDNA library according to the general protocol outlinedby Huse et al., supra. The general principles and methods of recombinantpolypeptide production discussed above are applicable for antibodyproduction by recombinant methods.

When desired, antibodies capable of specifically recognizing a mutantpeptide of the present invention can be tested for theircross-reactivity against the wild-type peptide and thus distinguishedfrom the antibodies against the wild-type protein. For instance,antisera obtained from an animal immunized with a mutant peptide can berun through a column on which a wild-type peptide is immobilized. Theportion of the antisera that passes through the column recognizes onlythe mutant peptide and not the wild-type peptide. Similarly, monoclonalantibodies against a mutant peptide can also be screened for theirexclusivity in recognizing only the mutant but not the wild-typepeptide.

Polyclonal or monoclonal antibodies that specifically recognize only themutant peptide of the present invention but not the wild-type peptideare useful for isolating the mutant protein from the wild-type protein,for example, by incubating a sample with a mutant peptide-specificpolyclonal or monoclonal antibody immobilized on a solid support.

Immunoassays for Detecting Mutant Peptide Expression

Once antibodies specific for a mutant peptide of the present inventionare available, the amount of the polypeptide in a sample, e.g., a celllysate, can be measured by a variety of immunoassay methods providingqualitative and quantitative results to a skilled artisan. For a reviewof immunological and immunoassay procedures in general see, e.g, Stites,supra; U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168.

Labeling in Immunoassays

Immunoassays often utilize a labeling agent to specifically bind to andlabel the binding complex formed by the antibody and the target protein.The labeling agent may itself be one of the moieties comprising theantibody/target protein complex, or may be a third moiety, such asanother antibody, that specifically binds to the antibody/target proteincomplex. A label may be detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Examples include, but are not limited to, magnetic beads (e.g.,Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texasred, rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase, andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads.

In some cases, the labeling agent is a second antibody bearing adetectable label. Alternatively, the second antibody may lack a label,but it may, in turn, be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond antibody can be modified with a detectable moiety, such asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G, can also be used as the labelagents. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally, Kronval, et al. J. Immunol., 111: 1401-1406 (1973); andAkerstrom, et al., J. Immunol., 135: 2589-2542 (1985)).

Immunoassay Formats

Immunoassays for detecting a target protein of interest (e.g., a mutanthuman growth hormone) from samples may be either competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of captured target protein is directly measured. In one preferred“sandwich” assay, for example, the antibody specific for the targetprotein can be bound directly to a solid substrate where the antibody isimmobilized. It then captures the target protein in test samples. Theantibody/target protein complex thus immobilized is then bound by alabeling agent, such as a second or third antibody bearing a label, asdescribed above.

In competitive assays, the amount of target protein in a sample ismeasured indirectly by measuring the amount of an added (exogenous)target protein displaced (or competed away) from an antibody specificfor the target protein by the target protein present in the sample. In atypical example of such an assay, the antibody is immobilized and theexogenous target protein is labeled. Since the amount of the exogenoustarget protein bound to the antibody is inversely proportional to theconcentration of the target protein present in the sample, the targetprotein level in the sample can thus be determined based on the amountof exogenous target protein bound to the antibody and thus immobilized.

In some cases, western blot (immunoblot) analysis is used to detect andquantify the presence of a mutant peptide in the samples. The techniquegenerally comprises separating sample proteins by gel electrophoresis onthe basis of molecular weight, transferring the separated proteins to asuitable solid support (such as a nitrocellulose filter, a nylon filter,or a derivatized nylon filter) and incubating the samples with theantibodies that specifically bind the target protein. These antibodiesmay be directly labeled or alternatively may be subsequently detectedusing labeled antibodies (e.g., labeled sheep anti-mouse antibodies)that specifically bind to the antibodies against a mutant peptide.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.,Amer. Clin. Prod. Rev., 5: 34-41 (1986)).

The Conjugates

In a representative aspect, the present invention provides aglycoconjugate between a peptide and a selected modifying group, inwhich the modifying group is conjugated to the peptide through aglycosyl linking group, e.g., an intact glycosyl linking group. Theglycosyl linking group is directly bound to an O-linked glycosylationsite on the peptide or, alternatively, it is bound to an O-linkedglycosylation site through one or more additional glycosyl residues.Methods of preparing the conjugates are set forth herein and in U.S.Pat. Nos. 5,876,980; 6,030,815; 5,728,554; 5,922,577; WO 98/31826;US2003180835; and WO 03/031464.

Exemplary peptides include an O-linked GalNAc residue that is bound tothe O-linked glycosylation site through the action of a GalNActransferase. The GalNAc itself may be the intact glycosyl linking group.The GalNAc may also be further elaborated by, for example, a Gal or Siaresidue, either of which can act as the intact glycosyl linking group.In representative embodiments, the O-linked saccharyl residue isGalNAc-X, GalNAc-Gal-Sia-X, or GalNAc-Gal-Gal-Sia-X, in which X is amodifying group.

In an exemplary embodiment, the peptide is a mutant peptide thatincludes an O-linked glycosylation site not present in the wild-typepeptide. The peptide is preferably O-glycosylated at the mutated sitewith a GalNAc residue. The discussion immediately preceding regardingthe structure of the saccharyl moiety is relevant here as well.

The link between the peptide and the selected moiety includes an intactglycosyl linking group interposed between the peptide and the modifyingmoiety. As discussed herein, the selected moiety is essentially anyspecies that can be attached to a saccharide unit, resulting in a“modified sugar” that is recognized by an appropriate transferaseenzyme, which appends the modified sugar onto the peptide. Thesaccharide component of the modified sugar, when interposed between thepeptide and a selected moiety, becomes an “intact glycosyl linkinggroup.” The glycosyl linking group is formed from any mono- oroligo-saccharide that, after modification with a selected moiety, is asubstrate for an appropriate transferase.

The conjugates of the invention will typically correspond to the generalstructure:

in which the symbols a, b, c, d and s represent a positive, non-zerointeger; and t is either 0 or a positive integer. The “agent” is atherapeutic agent, a bioactive agent, a detectable lable, water-solublemoiety or the like. The “agent” can be a peptide, e.g, enzyme, antibody,anitgen, etc. The linker can be any of a wide array of linking groups,infra. Alternatively, the linker may be a single bond or a “zero orderlinker.” The identity of the peptide is without limitation.

In an exemplary embodiment, the selected moiety is a water-solublepolymer, e.g., PEG, m-PEG, PPG, m-PPG, etc. The water-soluble polymer iscovalently attached to the peptide via a glycosyl linking group. Theglycosyl linking group is covalently attached to either an amino acidresidue or a glycosyl residue of the peptide. Alternatively, theglycosyl linking group is attached to one or more glycosyl units of aglycopeptide. The invention also provides conjugates in which theglycosyl linking group (e.g., GalNAc) is attached to an amino acidresidue (e.g., Thr or Ser).

In an exemplary embodiment, the protein is an interferon. Theinterferons are antiviral glycoproteins that, in humans, are secreted byhuman primary fibroblasts after induction with virus or double-strandedRNA. Interferons are of interest as therapeutics, e.g, antiviral agents(e.g., hepatitis B and C), antitumor agents (e.g., hepatocellularcarcinoma) and in the treatment of multiple sclerosis. For referencesrelevant to interferon-α, see, Asano, et al., Eur. J. Cancer, 27(Suppl4):S21-S25 (1991); Nagy, et al., Anticancer Research, 8(3):467-470(1988); Dron, et al., J. Biol. Regul. Homeost. Agents, 3(1):13-19(1989); Habib, et al., Am. Surg., 67(3):257-260 (3/2001); and Sugyiama,et al., Eur. J. Biochem., 217:921-927 (1993). For references discussingintefereon-β, see, e.g., Yu, et al., J Neuroimmunol., 64(1):91-100(1996); Schmidt, J., J. Neurosci. Res., 65(1):59-67 (2001); Wender, etal., Folia Neuropathol., 39(2):91-93 (2001); Martin, et al., SpringerSemin. Immunopathol., 18(1):1-24 (1996); Takane, et al., J. Pharmacol.Exp. Ther., 294(2):746-752 (2000); Sburlati, et al., Biotechnol. Prog.,14:189-192 (1998); Dodd, et al., Biochimica et Biophysica Acta,787:183-187 (1984); Edelbaum, et al., J. Interferon Res., 12:449-453(1992); Conradt, et al., J. Biol. Chem., 262(30):14600-14605 (1987);Civas, et al., Eur. J. Biochem., 173:311-316 (1988); Demolder, et al.,J. Biotechnol., 32:179-189 (1994); Sedmak, et al., J. Interferon Res.,9(Suppl 1):S61-S65 (1989); Kagawa, et al., J. Biol. Chem.,263(33):17508-17515 (1988); Hershenson, et al., U.S. Pat. No. 4,894,330;Jayaram, et al., J. Interferon Res., 3(2): 177-180 (1983); Menge, etal., Develop. Biol. Standard., 66:391-401 (1987); Vonk, et al., J.Interferon Res., 3(2):169-175 (1983); and Adolf, et al., J. InterferonRes., 10:255-267 (1990).

In an exemplary interferon conjugate, interferon alpha, e.g., interferonalpha 2β, is conjugated to a water soluble polymer through an intactglycosyl linker.

In a further exemplary embodiment, the invention provides a conjugate ofhuman Glucagon-like peptide-1 (GLP-1). GLP-1 is protein that haspleiotropic effects in the maintenance of glyceric control of theorganism. GLP-1 is released in response to the oral ingestion of food.GLP-1 appears to regulate plasma glucose levels by a variety ofmechanisms including the enhancement of glucose dependent insulinsecretion, stimulation of proinsulin gene expression, suppression ofglucagon release and gastric emptying, enhancement of insulinsensitivity, and increase of satiety (see e.g., Xiao et al. (2001Biochemistry 40:2860, and Perfetti, R. and Merkel, P. (2000) European J.of Endocrinology 143:717). GLP-1 is rapidly cleared from the body. Thehalf life of GLP-1 in vivo is about 5 minutes, with clearance completedwithin about 12-13 minutes. Even when administered subcutaneously, GLP-1is cleared from the circulation within 90 minutes (Perfetti, R. andMerkel, P. supra)

In addition to providing conjugates that are formed through anenzymatically added intact glycosyl linking group, the present inventionprovides conjugates that are highly homogenous in their substitutionpatterns. Using the methods of the invention, it is possible to formpeptide conjugates in which essentially all of the modified sugarmoieties across a population of conjugates of the invention are attachedto a structurally identical amino acid or glycosyl residue. Thus, in asecond aspect, the invention provides a peptide conjugate having apopulation of water-soluble polymer moieties, which are covalently boundto the peptide through an intact glycosyl linking group. In a preferredconjugate of the invention, essentially each member of the population isbound via the glycosyl linking group to a glycosyl residue of thepeptide, and each glycosyl residue of the peptide to which the glycosyllinking group is attached has the same structure.

Also provided is a peptide conjugate having a population ofwater-soluble polymer moieties covalently bound thereto through aglycosyl linking group. In a preferred embodiment, essentially everymember of the population of water soluble polymer moieties is bound toan amino acid residue of the peptide via an intact glycosyl linkinggroup, and each amino acid residue having an intact glycosyl linkinggroup attached thereto has the same structure.

The present invention also provides conjugates analogous to thosedescribed above in which the peptide is conjugated to a therapeuticmoiety, diagnostic moiety, targeting moiety, toxin moiety or the likevia a glycosyl linking group. Each of the above-recited moieties can bea small molecule, natural polymer (e.g., polypeptide) or syntheticpolymer.

The conjugates of the invention can include glycosyl linking groups thatare mono- or multi-valent (e.g., antennary structures). Thus, conjugatesof the invention include both species in which a selected moiety isattached to a peptide via a monovalent glycosyl linking group. Alsoincluded within the invention are conjugates in which more than oneselected moiety is attached to a peptide via a multivalent linkinggroup.

The Methods

In addition to the conjugates discussed above, the present inventionprovides methods for preparing these and other conjugates. Moreover, theinvention provides methods of preventing, curing or ameliorating adisease state by administering a conjugate of the invention to a subjectat risk of developing a disease or condition, (e.g., diabetes orobesity) or a subject that has the disease or condition.

Thus, the invention provides a method of forming a covalent conjugatebetween a selected moiety and a peptide. In exemplary embodiments, theconjugate is formed between a water-soluble polymer, a therapeuticmoiety, targeting moiety or a biomolecule, and a glycosylated ofnon-glycosylated peptide. The polymer, therapeutic moiety or biomoleculeis conjugated to the peptide via a glycosyl linking group, which isinterposed between, and covalently linked to both the peptide and themodifying group (e.g. water-soluble polymer). The method includescontacting the peptide with a mixture containing a modified sugar and aglycosyltransferase for which the modified sugar is a substrate. Thereaction is conducted under conditions appropriate to form a covalentbond between the modified sugar and the peptide. The sugar moiety of themodified sugar is preferably selected from nucleotide sugars, activatedsugars and sugars, which are neither nucleotides nor activated.

The acceptor peptide (O-glycosylated or non-glycosylated) is typicallysynthesized de novo, or recombinantly expressed in a prokaryotic cell(e.g., bacterial cell, such as E. coli) or in a eukaryotic cell such asa mammalian, yeast, insect, fungal or plant cell. The peptide can beeither a full-length protein or a fragment. Moreover, the peptide can bea wild type or mutated peptide. In an exemplary embodiment, the peptideincludes a mutation that adds one or more N- or O-linked glycosylationsites to the peptide sequence.

The method of the invention also provides for modification ofincompletely glycosylated peptides that are produced recombinantly. Manyrecombinantly produced glycoproteins are incompletely glycosylated,exposing carbohydrate residues that may have undesirable properties,e.g., immunogenicity, recognition by the RES. Employing a modified sugarin a method of the invention, the peptide can be simultaneously furtherglycosylated and derivatized with, e.g., a water-soluble polymer,therapeutic agent, or the like. The sugar moiety of the modified sugarcan be the residue that would properly be conjugated to the acceptor ina fully glycosylated peptide, or another sugar moiety with desirableproperties.

Any peptides modified by the methods of the invention. However, thepeptides are typically mutated peptides, produced by methods known inthe art, such as site-directed mutagenesis. Glycosylation of peptides istypically either N-linked or O-linked. An exemplary N-linkage is theattachment of the modified sugar to the side chain of an asparagineresidue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of a carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. O-linked glycosylation refers to the attachment ofone sugar (e.g., N-acetylgalactosamine, galactose, mannose, GlcNAc,glucose, fucose or xylose) to the hydroxy side chain of a hydroxyaminoacid, preferably serine or threonine, although unusual or non-naturalamino acids, e.g., 5-hydroxyproline or 5-hydroxylysine may also be used.

Moreover, in addition to peptides, the methods of the present inventioncan be practiced with other biological structures (e.g., glycolipids,lipids, sphingoids, ceramides, whole cells, and the like, containing anO-linked glycosylation site).

Addition of glycosylation sites to a peptide or other structure isconveniently accomplished by altering the amino acid sequence such thatit contains one or more glycosylation sites. The addition may also bemade by the incorporation of one or more species presenting an —OHgroup, preferably serine or threonine residues, within the sequence ofthe peptide (for O-linked glycosylation sites). The addition may be madeby mutation or by full chemical synthesis of the peptide. The peptideamino acid sequence is preferably altered through changes at the DNAlevel, particularly by mutating the DNA encoding the peptide atpreselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation(s) are preferably madeusing methods known in the art.

In an exemplary embodiment, the glycosylation site is added by shufflingpolynucleotides. Polynucleotides encoding a candidate peptide can bemodulated with DNA shuffling protocols. DNA shuffling is a process ofrecursive recombination and mutation, performed by random fragmentationof a pool of related genes, followed by reassembly of the fragments by apolymerase chain reaction-like process. See, e.g., Stemmer, Proc. Natl.Acad. Sci. USA 91:10747-10751 (1994); Stemmer, Nature 370:389-391(1994); and U.S. Pat. Nos. 5,605,793, 5,837,458, 5,830,721 and5,811,238.

The present invention also provides means of adding (or removing) one ormore selected glycosyl residues to a peptide, after which a modifiedsugar is conjugated to at least one of the selected glycosyl residues ofthe peptide. The present embodiment is useful, for example, when it isdesired to conjugate the modified sugar to a selected glycosyl residuethat is either not present on a peptide or is not present in a desiredamount. Thus, prior to coupling a modified sugar to a peptide, theselected glycosyl residue is conjugated to the peptide by enzymatic orchemical coupling. In another embodiment, the glycosylation pattern of aglycopeptide is altered prior to the conjugation of the modified sugarby the removal of a carbohydrate residue from the glycopeptide. See, forexample WO 98/31826.

Addition or removal of any carbohydrate moieties present on theglycopeptide is accomplished either chemically or enzymatically.Chemical deglycosylation is preferably brought about by exposure of thepolypeptide variant to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the peptide intact. Chemicaldeglycosylation is described by Hakimuddin et al., Arch. Biochem.Biophys. 259: 52 (1987) and by Edge et al., Anal. Biochem. 118: 131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptidevariants can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol. 138:350 (1987).

Chemical addition of glycosyl moieties is carried out by anyart-recognized method. Enzymatic addition of sugar moieties ispreferably achieved using a modification of the methods set forthherein, substituting native glycosyl units for the modified sugars usedin the invention. Other methods of adding sugar moieties are disclosedin U.S. Pat. Nos. 5,876,980, 6,030,815, 5,728,554, and 5,922,577.

Exemplary attachment points for selected glycosyl residue include, butare not limited to: (a) consensus sites for N-linked glycosylation, andsites for O-linked glycosylation; (b) terminal glycosyl moieties thatare acceptors for a glycosyltransferase; (c) arginine, asparagine andhistidine; (d) free carboxyl groups; (e) free sulfhydryl groups such asthose of cysteine; (f) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (g) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (h) the amide group ofglutamine. Exemplary methods of use in the present invention aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston, CRC CRIT. REV. BIOCHEM., pp. 259-306 (1981).

In one embodiment, the invention provides a method for linking two ormore peptides through a linking group. The linking group is of anyuseful structure and may be selected from straight- and branched-chainstructures. Preferably, each terminus of the linker, which is attachedto a peptide, includes a modified sugar (i.e., a nascent intact glycosyllinking group).

In an exemplary method of the invention, two peptides are linkedtogether via a linker moiety that includes a PEG linker. The constructconforms to the general structure set forth in the cartoon above. Asdescribed herein, the construct of the invention includes two intactglycosyl linking groups (i.e., s+t=1). The focus on a PEG linker thatincludes two glycosyl groups is for purposes of clarity and should notbe interpreted as limiting the identity of linker arms of use in thisembodiment of the invention.

Thus, a PEG moiety is functionalized at a first terminus with a firstglycosyl unit and at a second terminus with a second glycosyl unit. Thefirst and second glycosyl units are preferably substrates for differenttransferases, allowing orthogonal attachment of the first and secondpeptides to the first and second glycosylunits, respectively. Inpractice, the (glycosyl)¹-PEG-(glycosyl)² linker is contacted with thefirst peptide and a first transferase for which the first glycosyl unitis a substrate, thereby forming (peptide)¹-(glycosyl)¹-PEG-(glycosyl)².Transferase and/or unreacted peptide is then optionally removed from thereaction mixture. The second peptide and a second transferase for whichthe second glycosyl unit is a substrate are added to the(peptide)¹-(glycosyl)¹-PEG-(glycosyl)² conjugate, forming(peptide)¹-(glycosyl)¹-PEG-(glycosyl)²-(peptide)²; at least one of theglycosyl residues is either directly or indirectly O-linked. Those ofskill in the art will appreciate that the method outlined above is alsoapplicable to forming conjugates between more than two peptides by, forexample, the use of a branched PEG, dendrimer, poly(amino acid),polysaccharide or the like.

The use of reactive derivatives of PEG (or other linkers) to attach oneor more peptide moieties to a linker is within the scope of the presentinvention. The invention is not limited by the identity of the reactivePEG analogue. Many activated derivatives of poly(ethyleneglycol) areavailable commercially and in the literature. It is well within theabilities of one of skill to choose, and synthesize if necessary, anappropriate activated PEG derivative with which to prepare a substrateuseful in the present invention. See, Abuchowski et al. Cancer Biochem.Biophys., 7: 175-186 (1984); Abuchowski et al., J. Biol. Chem., 252:3582-3586 (1977); Jackson et al., Anal. Biochem., 165: 114-127 (1987);Koide et al., Biochem Biophys. Res. Commun., 111: 659-667 (1983)),tresylate (Nilsson et al., Methods Enzymol., 104: 56-69 (1984); Delgadoet al., Biotechnol. Appl. Biochem., 12: 119-128 (1990));N-hydroxysuccinimide derived active esters (Buckmann et al., Makromol.Chem., 182: 1379-1384 (1981); Joppich et al., Makromol. Chem., 180:1381-1384 (1979); Abuchowski et al., Cancer Biochem. Biophys., 7:175-186 (1984); Katre et al. Proc. Natl. Acad. Sci. USA., 84: 1487-1491(1987); Kitamura et al., Cancer Res., 51: 4310-4315 (1991); Boccu etal., Z. Naturforsch., 38C: 94-99 (1983), carbonates (Zalipsky et al.,POLY(ETHYLENE GLYCOL) CHEMISTRY: BIOTECHNICAL AND BIOMEDICALAPPLICATIONS, Harris, Ed., Plenum Press, New York, 1992, pp. 347-370;Zalipsky et al., Biotechnol. Appl. Biochem., 15: 100-114 (1992);Veronese et al., Appl. Biochem. Biotech., 11: 141-152 (1985)),imidazolyl formates (Beauchamp et al., Anal. Biochem., 131: 25-33(1983); Berger et al., Blood, 71: 1641-1647 (1988)), 4-dithiopyridines(Woghiren et al., Bioconjugate Chem., 4: 314-318 (1993)), isocyanates(Byun et al., ASAIO Journal, M649-M-653 (1992)) and epoxides (U.S. Pat.No. 4,806,595, issued to Noishiki et al., (1989). Other linking groupsinclude the urethane linkage between amino groups and activated PEG.See, Veronese, et al., Appl. Biochem. Biotechnol., 11: 141-152 (1985).

In another exemplary embodiment in which a reactive PEG derivative isutilized, the invention provides a method for extending theblood-circulation half-life of a selected peptide, in essence targetingthe peptide to the blood pool, by conjugating the peptide to a syntheticor natural polymer of a size sufficient to retard the filtration of theprotein by the glomerulus (e.g., albumin). For example, GLP-1 can beconjugated to albumin via a PEG linker using a combination of chemicaland enzymatic modification.

Modified Sugars

Modified glycosyl donor species (“modified sugars”) are preferablyselected from modified sugar nucleotides, activated modified sugars andmodified sugars that are simple saccharides that are neither nucleotidesnor activated. Any desired carbohydrate structure can be added to apeptide using the methods of the invention. Typically, the structurewill be a monosaccharide, but the present invention is not limited tothe use of modified monosaccharide sugars; oligosaccharides andpolysaccharides are useful as well.

The modifying group is attached to a sugar moiety by enzymatic means,chemical means or a combination thereof, thereby producing a modifiedsugar. The sugars are substituted at any position that allows for theattachment of the modifying moiety, yet which still allows the sugar tofunction as a substrate for the enzyme used to ligate the modified sugarto the peptide. In a preferred embodiment, when sialic acid is thesugar, the sialic acid is substituted with the modifying group at eitherthe 9-position on the pyruvyl side chain or at the 5-position on theamine moiety that is normally acetylated in sialic acid.

In certain embodiments of the present invention, a modified sugarnucleotide is utilized to add the modified sugar to the peptide.Exemplary sugar nucleotides that are used in the present invention intheir modified form include nucleotide mono-, di- or triphosphates oranalogs thereof. In a preferred embodiment, the modified sugarnucleotide is selected from a UDP-glycoside, CMP-glycoside, or aGDP-glycoside. Even more preferably, the modified sugar nucleotide isselected from an UDP-galactose, UDP-galactosamine, UDP-glucose,UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid, or CMP-NeuAc.N-acetylamine derivatives of the sugar nucletides are also of use in themethod of the invention.

The invention also provides methods for synthesizing a modified peptideusing a modified sugar, e.g., modified-galactose, -fucose, -GalNAc and-sialic acid. When a modified sialic acid is used, either asialyltransferase or a trans-sialidase (for α2,3-linked sialic acidonly) can be used in these methods.

In other embodiments, the modified sugar is an activated sugar.Activated modified sugars, which are useful in the present invention aretypically glycosides which have been synthetically altered to include anactivated leaving group. As used herein, the term “activated leavinggroup” refers to those moieties, which are easily displaced inenzyme-regulated nucleophilic substitution reactions. Many activatedsugars are known in the art. See, for example, Vocadlo et al., InCARBOHYDRATE CHEMISTRY AND BIOLOGY, Vol. 2, Ernst et al. Ed., Wiley-VCHVerlag: Weinheim, Germany, 2000; Kodama et al., Tetrahedron Lett. 34:6419 (1993); Lougheed, et al., J. Biol. Chem. 274: 37717 (1999)).

Examples of activating groups (leaving groups) include fluoro, chloro,bromo, tosylate ester, mesylate ester, triflate ester and the like.Preferred activated leaving groups, for use in the present invention,are those that do not significantly sterically encumber the enzymatictransfer of the glycoside to the acceptor. Accordingly, preferredembodiments of activated glycoside derivatives include glycosylfluorides and glycosyl mesylates, with glycosyl fluorides beingparticularly preferred. Among the glycosyl fluorides, α-galactosylfluoride, α-mannosyl fluoride, α-glucosyl fluoride, α-fucosyl fluoride,α-xylosyl fluoride, α-sialyl fluoride, α-N-acetylglucosaminyl fluoride,α-N-acetylgalactosaminyl fluoride, β-galactosyl fluoride, β-mannosylfluoride, β-glucosyl fluoride, β-fucosyl fluoride, β-xylosyl fluoride,β-sialyl fluoride, β-N-acetylglucosaminyl fluoride andβ-N-acetylgalactosaminyl fluoride are most preferred.

By way of illustration, glycosyl fluorides can be prepared from the freesugar by first acetylating the sugar and then treating it withHF/pyridine. This generates the thermodynamically most stable anomer ofthe protected (acetylated) glycosyl fluoride (i.e., the α-glycosylfluoride). If the less stable anomer (i.e., the β-glycosyl fluoride) isdesired, it can be prepared by converting the peracetylated sugar withHBr/HOAc or with HCl to generate the anomeric bromide or chloride. Thisintermediate is reacted with a fluoride salt such as silver fluoride togenerate the glycosyl fluoride. Acetylated glycosyl fluorides may bedeprotected by reaction with mild (catalytic) base in methanol (e.g.NaOMe/MeOH). In addition, many glycosyl fluorides are commerciallyavailable.

Other activated glycosyl derivatives can be prepared using conventionalmethods known to those of skill in the art. For example, glycosylmesylates can be prepared by treatment of the fully benzylatedhemiacetal form of the sugar with mesyl chloride, followed by catalytichydrogenation to remove the benzyl groups.

In a further exemplary embodiment, the modified sugar is anoligosaccharide having an antennary structure. In a preferredembodiment, one or more of the termini of the antennae bear themodifying moiety. When more than one modifying moiety is attached to anoligosaccharide having an antennary structure, the oligosaccharide isuseful to “amplify” the modifying moiety; each oligosaccharide unitconjugated to the peptide attaches multiple copies of the modifyinggroup to the peptide. The general structure of a typical conjugate ofthe invention as set forth in the drawing above, encompasses multivalentspecies resulting from preparing a conjugate of the invention utilizingan antennary structure. Many antennary saccharide structures are knownin the art, and the present method can be practiced with them withoutlimitation.

Exemplary modifying groups are discussed below. The modifying groups canbe selected for their ability to impart to a peptide one or moredesirable property. Exemplary properties include, but are not limitedto, enhanced pharmacokinetics, enhanced pharmacodynamics, improvedbiodistribution, providing a polyvalent species, improved watersolubility, enhanced or diminished lipophilicity, and tissue targeting.

Water-Soluble Polymers

The hydrophilicity of a selected peptide is enhanced by conjugation withpolar molecules such as amine-, ester-, hydroxyl- andpolyhydroxyl-containing molecules. Representative examples include, butare not limited to, polylysine, polyethyleneimine, and polyethers, e.g.,poly(ethyleneglycol), m-poly(ethylene glycol), poly(propyleneglycol),m-poly(ethylene glycol), and other O-alkyl poly(alkylene glycol)moieties. Preferred water-soluble polymers are essentiallynon-fluorescent, or emit such a minimal amount of fluorescence that theyare inappropriate for use as a fluorescent marker in an assay. Moreover,it is generally preferred to use polymers that are not naturallyoccurring sugars. An exception to this preference is the use of anotherwise naturally occurring sugar that is modified by covalentattachment of another entity (e.g., poly(ethylene glycol),poly(propylene glycol), biomolecule, therapeutic moiety, diagnosticmoiety, etc.). In another exemplary embodiment, a therapeutic sugarmoiety is conjugated to a linker arm and the sugar-linker arm cassetteis subsequently conjugated to a peptide via a method of the invention.

Methods and chemistry for activation of water-soluble polymers andsaccharides as well as methods for conjugating saccharides and polymersto various species are described in the literature. Commonly usedmethods for activation of polymers include activation of functionalgroups with cyanogen bromide, periodate, glutaraldehyde, biepoxides,epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides,trichlorotriazine, etc. (see, R. F. Taylor, (1991), PROTEINIMMOBILISATION. FUNDAMENTALS AND APPLICATIONS, Marcel Dekker, N.Y.; S.S. Wong, (1992), CHEMISTRY OF PROTEIN CONJUGATION AND CROSSLINKING, CRCPress, Boca Raton; G. T. Hermanson et al., (1993), IMMOBILIZED AFFINITYLIGAND TECHNIQUES, Academic Press, N.Y.; Dunn, R. L., et al., Eds.POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol.469, American Chemical Society, Washington, D.C. 1991).

Many water-soluble polymers are known to those of skill in the art andare useful in practicing the present invention. The term water-solublepolymer encompasses species such as saccharides (e.g., dextran, amylose,hyalouronic acid, poly(sialic acid), heparans, heparins, etc.); poly(amino acids); nucleic acids; synthetic polymers (e.g., poly(acrylicacid), poly(ethers), e.g., poly(ethylene glycol); peptides, proteins,and the like. The present invention may be practiced with anywater-soluble polymer with the sole limitation that the polymer mustinclude a point at which the remainder of the conjugate can be attached.

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and more WO 93/15189, and for conjugation between activatedpolymers and peptides, e.g. Coagulation Factor VIII (WO 94/15625),haemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No.4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App.Biochem. Biotech. 11: 141-45 (1985)).

Preferred water-soluble polymers are those in which a substantialproportion of the polymer molecules in a sample of the polymer are ofapproximately the same molecular weight; such polymers are“homodisperse.”

The present invention is further illustrated by reference to apoly(ethylene glycol) or monomethoxy-poly(ethylene glycol) (m-PEG)conjugate. Several reviews and monographs on the functionalization andconjugation of PEG are available. See, for example, Harris, Macronol.Chem. Phys. C25: 325-373 (1985); Scouten, Methods in Enzymology 135:30-65 (1987); Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992);Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9:249-304 (1992); Zalipsky, Bioconjugate Chem. 6: 150-165 (1995); andBhadra, et al., Pharmazie, 57:5-29 (2002).

The poly(ethylene glycol) useful in forming the conjugate of theinvention is either linear or branched.

The in vivo half-life of therapeutic glycopeptides can also be enhancedwith water-soluble polymers such as polyethylene glycol (PEG, m-PEG) andpolypropylene glycol (PPG). For example, chemical modification ofproteins with PEG (PEG-ylation, m-PEG-ylation) increases their molecularsize and decreases their surface- and functional group-accessibility,each of which are dependent on the size of the PEG attached to theprotein. This results in an improvement of plasma half-lives and inproteolytic-stability, and a decrease in immunogenicity and hepaticuptake (Chaffee et al. J. Clin. Invest. 89: 1643-1651 (1992); Pyatak etal. Res. Commun. Chem. Pathol Pharmacol. 29: 113-127 (1980)). PEGylationof interleukin-2 has been reported to increase its antitumor potency invivo (Katre et al. Proc. Natl. Acad. Sci. USA. 84: 1487-1491 (1987)) andPEG-ylation of a F(ab′)2 derived from the monoclonal antibody A7 hasimproved its tumor localization (Kitamura et al. Biochem. Biophys. Res.Commun. 28: 1387-1394 (1990)). Thus, in another preferred embodiment,the in vivo half-life of a peptide such as e.g., Glucagon-likepeptide-1, derivatized with a water-soluble polymer by a method of theinvention is increased relevant to the in vivo half-life of thenon-derivatized peptide.

The increase in peptide in vivo half-life is best expressed as a rangeof percent increase in this quantity. The lower end of the range ofpercent increase is about 40%, about 60%, about 80%, about 100%, about150% or about 200%. The upper end of the range is about 60%, about 80%,about 100%, about 150%, or more than about 250%.

In selected glyco-PEG-ylated peptides of the invention, the PEG-intactglycosyl linker cassette has the structure:

in which L is a substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl linker moiety joining the sialic acid moietyand the PEG moiety. The index n is selected from the integers from 0 toabout 2500, more preferably from about 50 to about 1500, and morepreferable still from about 100 to about 600. An example of thisstructure has the formula:

in which the index “s” represents an integer from 0 to 20.

PEG moieties of any molecular weight, e.g., 5 Kd, 10 Kd, 20 Kd, 30 kD,40 kD, 60 kD and 100 kD are of use in the present invention.

Exemplary activated modified sugars of use in preparing water-solublepolymer-peptide conjugates of the invention include; an linear PEGspecies (A) and a branched PEG species (B):

Following their conjugation to an O-linked site, exemplary PEG-sialicacid-glycosyl moieties can have one or more of the following structures:

In an exemplary embodiment, the Thr shown in the structures above isThr¹⁰⁶ of interferon alpha 2b.

In another exemplary embodiment, poly(ethylene glycol) molecules of usein the invention include, but are not limited to, those species setforth below.

in which R² is H, substituted or unsubstituted alkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted heterocycloalkyl, substituted or unsubstitutedheteroalkyl, e.g., acetal, OHC—, H₂N—(CH₂)_(q)—, HS—(CH₂)_(q), and—(CH₂)_(q)C(Y¹)Z²; -sugar-nucleotide, or protein. The index “n”represents an integer from 1 to 2500. The indeces m, o, and qindependently represent integers from 0 to 20. The symbols Z¹ and Z²independently represent OH, NH₂, halogen, S—R³, the alcohol portion ofactivated esters, —(CH₂)_(p)C(Y²)V, —(CH₂)_(p)U(CH₂)_(s)C(Y²)_(v),sugar-nucleotide, protein, and leaving groups, e.g., imidazole,p-nitrophenyl, HOBT, tetrazole, halide. The symbols X, Y¹, Y², A¹, and Uindependently represent the moieties O, S, N—R⁴. The symbol V representsOH, NH₂, halogen, S—R⁵, the alcohol component of activated esters, theamine component of activated amides, sugar-nucleotides, and proteins.The indeces p, q, s and v are members independently selected from theintegers from 0 to 20. The symbols R³, R⁴ and R⁵ independently representH, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heterocycloalkyl and substituted or unsubstitutedheteroaryl.

In other exemplary embodiments, the poly(ethylene glycol) molecule isselected from the following:

In an exemplary embodiment, the invention provides a glycopeptide thatis conjugated to a polymeric modifying moiety through an intact glycosyllinking group having a formula that is selected from:

In Formulae I R² is H, CH₂OR⁷, COOR⁷ or OR⁷, in which R⁷ represents H,substituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl. When COOR⁷ is a carboxylic acid or carboxylate, both formsare represented by the designation of the single structure COO⁻ or COOH.In Formulae I and II, the symbols R³, R⁴, R⁵, R⁶ and R^(6′)independently represent H, substituted or unsubstituted alkyl, OR⁸,NHC(O)R⁹. The index d is 0 or 1. R⁸ and R⁹ are independently selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, sialic acid or polysialic acid. At least one of R³, R⁴, R⁵,R⁶ or R^(6′)includes the polymeric modifying moiety e.g., PEG, linkedthrough a bond or a linking group. In an exemplary embodiment, R⁶ andR^(6′), together with the carbon to which they are attached arecomponents of the pyruvyl side chain of sialic acid. In a furtherexemplary embodiment, this side chain is functionalized with thepolymeric modifying moiety. In another exemplary embodiment, R⁶ andR^(6′), together with the carbon to which they are attached arecomponents of the side chain of sialic acid and the polymeric modifyingmoiety is a component of R⁵.

In a further exemplary embodiment, the polymeric modifying moiety isbound to the sugar core, generally through a heteroatom, e.g, nitrogen,on the core through a linker, L, as shown below:

R¹ is the polymeric moiety and L is selected from a bond and a linkinggroup. The index w represents an integer selected from 1-6, preferably1-3 and more preferably 1-2. Exemplary linking groups includesubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl moieties and sialic acid. An exemplary component of thelinker is an acyl moiety.

An exemplary compound according to the invention has a structureaccording to Formulae I or II, in which at least one of R², R³, R⁴, R⁵,R⁶ or R^(6′) has the formula:

In another example according to this embodiment at least one of R², R³,R⁴, R⁵, R⁶ or R^(6′) has the formula:

in which s is an integer from 0 to 20 and R¹ is a linear polymericmodifying moiety.

In an exemplary embodiment, the polymeric modifying moiety-linkerconstruct is a branched structure that includes two or more polymericchains attached to the central moiety. In this embodiment, the constructhas the formula:

in which R¹ and L are as discussed above and w′ is an integer from 2 to6, preferably from 2 to 4 and more preferably from 2 to 3.

When L is a bond it is formed between a reactive functional group on aprecursor of R¹ and a reactive functional group of complementaryreactivity on the saccharyl core. When L is a non-zero order linker, aprecursor of L can be in place on the glycosyl moiety prior to reactionwith the R¹ precursor. Alternatively, the precursors of R¹ and L can beincorporated into a preformed cassette that is subsequently attached tothe glycosyl moiety. As set forth herein, the selection and preparationof precursors with appropriate reactive functional groups is within theability of those skilled in the art. Moreover, coupling the precursorsproceeds by chemistry that is well understood in the art.

In an exemplary embodiment, L is a linking group that is formed from anamino acid, or small peptide (e.g., 1-4 amino acid residues) providing amodified sugar in which the polymeric modifying moiety is attachedthrough a substituted alkyl linker. Exemplary linkers include glycine,lysine, serine and cysteine. The PEG moiety can be attached to the aminemoiety of the linker through an amide or urethane bond. The PEG islinked to the sulfur or oxygen atoms of cysteine and serine throughthioether or ether bonds, respectively.

In an exemplary embodiment, R⁵ includes the polymeric modifying moiety.In another exemplary embodiment, R⁵ includes both the polymericmodifying moiety and a linker, L, joining the modifying moiety to theremainder of the molecule. As discussed above, L can be a linear orbranched structure. Similarly, the polymeric modifying can be branchedor linear.

In one embodiment, the present invention provides an GLP-1 peptidecomprising the moiety:

wherein D is a member selected from —OH and R¹-L-HN—; G is a memberselected from H and R′-L- and —C(O)(C₁-C₆)alkyl; R¹ is a moietycomprising a straight-chain or branched poly(ethylene glycol) residue;and L is a linker, e.g., a bond (“zero order”), substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl. Inexemplary embodiments, when D is OH, G is R¹-L-, and when G is—C(O)(C₁-C₆)alkyl, D is R¹-L-NH—.

In another exemplary embodiment, the invention provides a conjugateformed between a modified sugar of the invention and a substrate GLP-1peptide. In this embodiment, the sugar moiety of the modified sugarbecomes a glycosyl linking group interposed between the peptidesubstrate and the modifying group. An exemplary glycosyl linking groupis an intact glycosyl linking group, in which the glycosyl moiety ormoieties forming the linking group are not degraded by chemical (e.g.,sodium metaperiodate) or enzymatic (e.g., oxidase) processes. Selectedconjugates of the invention include a modifying group that is attachedto the amine moiety of an amino-saccharide, e.g., mannosamine,glucosamine, galactosamine, sialic acid etc. Exemplary modifyinggroup-intact glycosyl linking group cassettes according to this motifare based on a sialic acid structure, such as those having the formulae:

In the formulae above, R¹ and L are as described above. Further detailabout the structure of exemplary R¹ groups is provided below.

In still a further exemplary embodiment, the conjugate is formed betweena substrate GLP-1 and a saccharyl moiety in which the modifying group isattached through a linker at the 6-carbon position of the saccharylmoiety. Thus, illustrative conjugates according to this embodiment havethe formula:

in which the radicals are as discussed above. Such saccharyl moietiesinclude, without limitation, glucose, glucosamine, N-acetyl-glucosamine,galactose, galactosamine, N-acetyl-galactosamine, mannose, mannosamine,N-acetyl-mannosamine, and the like.

Due to the versatility of the methods available for modifying glycosylresidues on a therapeutic peptide such as GLP-1, the glycosyl structureson the peptide conjugates of the invention can have substantially anystructure. Moreover, the glycans can be O-linked or N-linked. Asexemplified in the discussion below, each of the pyranose and furanosederivatives discussed above can be a component of a glycosyl moiety of apeptide.

The invention provides a modified GLP-1 peptide that includes a glycosylgroup having the formula:

In other embodiments, the group has the formula:

in which the index t is 0 or 1.

In a still further exemplary embodiment, the group has the formula:

in which the index t is 0 or 1.

In yet another embodiment, the group has the formula:

in which the index p represents and integer from 1 to 10; and a iseither 0 or 1.

In an exemplary embodiment, a glycoPEGylated GLP-1 peptide of theinvention includes at least one N-linked glycosyl residue selected fromthe glycosyl residues set forth below:

In the formulae above, the index t is 0 or 1 and the index p is aninteger from 1 to 10. The symbol R^(15′) represents H, OH (e.g.,Gal-OH), a sialyl moiety, a polymer modified sialyl moiety (i.e.,glycosyl linking group-polymeric modifying moiety (Sia-L-R¹)) or asialyl moiety to which is bound a polymer modified sialyl moiety (e.g.,Sia-Sia-L-R¹) (“Sia-Sia^(p)”). Exemplary polymer modified saccharylmoieties have a structure according to Formulae I and II. An exemplaryGLP-1 peptide of the invention will include at least one glycan having aR^(15′) that includes a structure according to Formulae I or II. Theoxygen, with the open valence, of Formulae I and II is preferablyattached through a glycosidic linkage to a carbon of a Gal or GalNAcmoiety. In a further exemplary embodiment, the oxygen is attached to thecarbon at position 3 of a galactose residue. In an exemplary embodiment,the modified sialic acid is linked α2,3-to the galactose residue. Inanother exemplary embodiment, the sialic acid is linked α2,6-to thegalactose residue.

In another exemplary embodiment, the invention provides a GLP-1 peptideconjugate that includes a glycosyl linking group, such as those setforth above, that is covalently attached to an amino acid residue of thepeptide. In one embodiment according to this motif, the glycosyl linkingmoiety is linked to a galactose residue through a Sia residue:

An exemplary species according to this motif is prepared by conjugatingSia-L-R¹ to a terminal sialic acid of a glycan using an enzyme thatforms Sia-Sia bonds, e.g., CST-II, ST8Sia-II, ST8Sia-III and ST8Sia-IV.

In another exemplary embodiment, the glycans have a formula that isselected from the group:

and combinations thereof.

The glycans of this group generally correspond to those found on a GLP-1peptide that is produced by insect (e.g., Sf-9) cells, followingremodeling according to the methods set forth herein. For exampleinsect-derived GLP-1 that is expressed with a tri-mannosyl core issubsequently contacted with a GlcNAc donor and a GlcNAc transferase anda Gal donor and a Gal transferase. Appending GlcNAc and Gal to thetri-mannosyl core is accomplished in either two steps or a single step.A modified sialic acid is added to at least one branch of the glycosylmoiety as discussed herein. Those Gal moieties that are notfunctionalized with the modified sialic acid are optionally “capped” byreaction with a sialic acid donor in the presence of a sialyltransferase.

In an exemplary embodiment, at least 60% of terminal Gal moieties in apopulation of peptides is capped with sialic acid, preferably at least70%, more preferably, at least 80%, still more preferably at least 90%and even more preferably at least 95%, 96%, 97%, 98% or 99% are cappedwith sialic acid.

In each of the formulae above, R^(15′) is as discussed above. Moreover,an exemplary modified GLP-1 peptide of the invention will include atleast one glycan with an R¹⁵ moiety having a structure according toFormulae I or II.

In an exemplary embodiment, the glycosyl linking moiety has the formula:

in which b is 0 or 1. The index s represents and integer from 1 to 10;and f represents and integer from 1 to 2500. Generally preferred is theuse of a PEG moiety that has a molecular weight of about 20 kDa.

In another exemplary embodiment, the GLP-1 is derived from insect cells,remodeled by adding GlcNAc and Gal to the mannose core andglycopegylated using a sialic acid bearing a linear PEG moiety,affording a GLP-1 peptide that comprises at least one moiety having theformula:

in which s represents and integer from 1 to 10; and f represents andinteger from 1 to 2500.

As discussed herein, the PEG of use in the conjugates of the inventioncan be linear or branched. An exemplary precursor of use to form thebranched conjugates according to this embodiment of the invention hasthe formula:

The branched polymer species according to this formula are essentiallypure water-soluble polymers. X^(3′) is a moiety that includes anionizable, e.g., OH, COOH, H₂PO₄, HSO₃, HPO₃, and salts thereof, etc.)or other reactive functional group, e.g., infra. C is carbon. X⁵ ispreferably a non-reactive group (e.g., H, unsubstituted alkyl,unsubstituted heteroalkyl), and can be a polymeric arm. R¹⁶ and R¹⁷ areindependently selected polymeric arms, e.g., nonpeptidic, nonreactivepolymeric arms (e.g., PEG)). X² and X⁴ are linkage fragments that arepreferably essentially non-reactive under physiological conditions,which may be the same or different. An exemplary linker includes neitheraromatic nor ester moieties. Alternatively, these linkages can includeone or more moiety that is designed to degrade under physiologicallyrelevant conditions, e.g., esters, disulfides, etc. X² and X⁴ joinpolymeric arms R¹⁶ and R¹⁷ to C. When X^(3′) is reacted with a reactivefunctional group of complementary reactivity on a linker, sugar orlinker-sugar cassette, X^(3′) is converted to a component of linkagefragment X³.

Exemplary linkage fragments for X², X³ and X⁴ are independently selectedand include S, SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH andNHC(O)O, and OC(O)NH, CH₂S, CH₂O, CH₂CH₂O, CH₂CH₂S, (CH₂)_(o)O,(CH₂)_(o)S or (CH₂)_(o)Y′-PEG wherein, Y′ is S, NH, NHC(O), C(O)NH,NHC(O)O, OC(O)NH, or O and o is an integer from 1 to 50. In an exemplaryembodiment, the linkage fragments X² and X⁴ are different linkagefragments.

In an exemplary embodiment, the precursor (III), or an activatedderivative thereof, is reacted with, and thereby bound to a sugar, anactivated sugar or a sugar nucleotide through a reaction between X^(3′)and a group of complementary reactivity on the sugar moiety, e.g., anamine. Alternatively, X^(3′) reacts with a reactive functional group ona precursor to linker, L. One or more of R², R³, R⁴, R⁵, R⁶ or R^(6′) ofFormulae I and II can include the branched polymeric modifying moiety,or this moiety bound through L.

In an exemplary embodiment, the moiety:

is the linker arm, L. In this embodiment, an exemplary linker is derivedfrom a natural or unnatural amino acid, amino acid analogue or aminoacid mimetic, or a small peptide formed from one or more such species.For example, certain branched polymers found in the compounds of theinvention have the formula:

X^(a) is a linkage fragment that is formed by the reaction of a reactivefunctional group, e.g., X^(3′), on a precursor of the branched polymericmodifying moiety and a reactive functional group on the sugar moiety, ora precursor to a linker. For example, when X^(3′) is a carboxylic acid,it can be activated and bound directly to an amine group pendent from anamino-saccharide (e.g., Sia, GalNH₂, GlcNH₂, ManNH₂, etc.), forming anX^(a) that is an amide. Additional exemplary reactive functional groupsand activated precursors are described hereinbelow. The index crepresents an integer from 1 to 10. The other symbols have the sameidentity as those discussed above.

In another exemplary embodiment, X^(a) is a linking moiety formed withanother linker:

in which X^(b) is a second linkage fragment and is independentlyselected from those groups set forth for X^(a), and, similar to L, L¹ isa bond, substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl.

Exemplary species for X^(a) and X^(b) include S, SC(O)NH, HNC(O)S,SC(O)O, O, NH, NHC(O), C(O)NH and NHC(O)O, and OC(O)NH.

In another exemplary embodiment, X⁴ is a peptide bond to R¹⁷, which isan amino acid, di-peptide (e.g., Lys-Lys) or tri-peptide (E.G.,Lys-Lys-Lys) in which the alpha-amine moiety(ies) and/or side chainheteroatom(s) are modified with a polymeric modifying moiety.

In a further exemplary embodiment, the conjugates of the inventioninclude a moiety, e.g., an R¹⁵ moiety that has a formula that isselected from:

in which the identity of the radicals represented by the various symbolsis the same as that discussed hereinabove. L^(a) is a bond or a linkeras discussed above for L and L′, e.g., substituted or unsubstitutedalkyl or substituted or unsubstituted heteroalkyl moiety. In anexemplary embodiment, L^(a) is a moiety of the side chain of sialic acidthat is functionalized with the polymeric modifying moiety as shown.Exemplary L^(a) moieties include substituted or unsubstituted alkylchains that include one or more OH or NH₂.

In yet another exemplary embodiment, the invention provides conjugateshaving a moiety, e.g., an R¹⁵ moiety with formula:

The identity of the radicals represented by the various symbols is thesame as that discussed hereinabove. As those of skill will appreciate,the linker arm in Formulae VI and VII is equally applicable to othermodified sugars set forth herein. In exemplary embodiment, the speciesof Formulae VI and VII are the R¹⁵ moieties attached to the glycanstructures set forth herein.

In yet another exemplary embodiment, the GLP-1 peptide includes an R¹⁵moiety with the formula:

in which the identities of the radicals are as discussed above. Anexemplary species for L^(a) is —(CH₂)_(j)C(O)NH(CH₂)_(h)C(O)NH—, inwhich h and j are independently selected integers from 0 to 10. Afurther exemplary species is —C(O)NH—.

The embodiments of the invention set forth above are further exemplifiedby reference to species in which the polymer is a water-soluble polymer,particularly poly(ethylene glycol) (“PEG”), e.g., methoxy-poly(ethyleneglycol). Those of skill will appreciate that the focus in the sectionsthat follow is for clarity of illustration and the various motifs setforth using PEG as an exemplary polymer are equally applicable tospecies in which a polymer other than PEG is utilized.

PEG of any molecular weight, e.g., 1 kDa, 2 kDa, 5 kDa, 10 kDa, 15 kDa,20 kDa, 30 kDa and 40 kDa is of use in the present invention.

In an exemplary embodiment, the R¹⁵ moiety has a formula that is amember selected from the group:

In each of the structures above, the linker fragment —NH(CH₂)_(a)— canbe present or absent.

In other exemplary embodiments, the conjugate includes an R¹⁵ moietyselected from the group:

In each of the formulae above, the indices e and f are independentlyselected from the integers from 1 to 2500. In further exemplaryembodiments, e and f are selected to provide a PEG moiety that is about1 kD, 2 kD, 10 kD, 15 kD, 20 kD, 30 kD or 40 kD. The symbol Q representssubstituted or unsubstituted alkyl (e.g., C₁-C₆ alkyl, e.g., methyl),substituted or unsubstituted heteroalkyl or H.

Other branched polymers have structures based on di-lysine (Lys-Lys)peptides, e.g.:

and tri-lysine peptides (Lys-Lys-Lys), e.g.:

In each of the figures above, e, f, f′ and f″ represent integersindependently selected from 1 to 2500. The indices q, q′ and q″represent integers independently selected from 1 to 20.

In another exemplary embodiment, the GLP-1 peptide comprises a glycosylmoiety selected from the formulae:

in which L^(a) is a bond or a linker as described herein; the index trepresents 0 or 1; and the index a represents 0 or 1. Each of thesegroups can be included as components of the mono-, bi-, tri- andtetra-antennary saccharide structures set forth above.

In yet another embodiment, the conjugates of the invention include amodified glycosyl residue that includes the substructure selected from:

in which the index a and the linker L^(a) are as discussed above. Theindex p is an integer from 1 to 10. The indices t and a areindependently selected from 0 or 1. Each of these groups can be includedas components of the mono-, bi-, tri- and tetra-antennary saccharidestructures set forth above.

In a further exemplary embodiment, the invention utilizes modifiedsugars in which the 6-hydroxyl position is converted to thecorresponding amine moiety, which bears a linker-modifying groupcassette such as those set forth above. Exemplary saccharyl groups thatcan be used as the core of these modified sugars include Gal, GalNAc,Glc, GlcNAc, Fuc, Xyl, Man, and the like. A representative modifiedsugar according to this embodiment has the formula:

in which R¹¹-R¹⁴ are members independently selected from H, OH, C(O)CH₃,NH, and NH C(O)CH₃. R¹⁰ is a link to another glycosyl residue(—O-glycosyl) or to an amino acid of the GLP-1 peptide (—NH-(GLP-1)).R¹⁴ is OR¹, NHR¹ or NH-L-R¹. R¹ and NH-L-R¹ are as described above.

Selected conjugates according to this motif are based on mannose,galactose or glucose, or on species having the stereochemistry ofmannose, galactose or glucose. The general formulae of these conjugatesare:

As discussed above, the invention provides saccharides bearing amodifying group, activated analogues of these species and conjugatesformed between species such as peptides and lipids and a modifiedsaccharide of the invention.

Biomolecules

In another preferred embodiment, the modified sugar bears a biomolecule.In still further preferred embodiments, the biomolecule is a functionalprotein, enzyme, antigen, antibody, peptide, nucleic acid (e.g., singlenucleotides or nucleosides, oligonucleotides, polynucleotides andsingle- and higher-stranded nucleic acids), lectin, receptor or acombination thereof.

Preferred biomolecules are essentially non-fluorescent, or emit such aminimal amount of fluorescence that they are inappropriate for use as afluorescent marker in an assay. Moreover, it is generally preferred touse biomolecules that are not sugars. An exception to this preference isthe use of an otherwise naturally occurring sugar that is modified bycovalent attachment of another entity (e.g., PEG, biomolecule,therapeutic moiety, diagnostic moiety, etc.). In an exemplaryembodiment, a sugar moiety, which is a biomolecule, is conjugated to alinker arm and the sugar-linker arm cassette is subsequently conjugatedto a peptide via a method of the invention.

Biomolecules useful in practicing the present invention can be derivedfrom any source. The biomolecules can be isolated from natural sourcesor they can be produced by synthetic methods. Peptides can be naturalpeptides or mutated peptides. Mutations can be effected by chemicalmutagenesis, site-directed mutagenesis or other means of inducingmutations known to those of skill in the art. Peptides useful inpracticing the instant invention include, for example, enzymes,antigens, antibodies and receptors. Antibodies can be either polyclonalor monoclonal; either intact or fragments. The peptides are optionallythe products of a program of directed evolution

Both naturally derived and synthetic peptides and nucleic acids are ofuse in conjunction with the present invention; these molecules can beattached to a sugar residue component or a crosslinking agent by anyavailable reactive group. For example, peptides can be attached througha reactive amine, carboxyl, sulfhydryl, or hydroxyl group. The reactivegroup can reside at a peptide terminus or at a site internal to thepeptide chain. Nucleic acids can be attached through a reactive group ona base (e.g., exocyclic amine) or an available hydroxyl group on a sugarmoiety (e.g., 3′- or 5′-hydroxyl). The peptide and nucleic acid chainscan be further derivatized at one or more sites to allow for theattachment of appropriate reactive groups onto the chain. See, Chriseyet al. Nucleic Acids Res. 24: 3031-3039 (1996).

In a further preferred embodiment, the biomolecule is selected to directthe peptide modified by the methods of the invention to a specifictissue, thereby enhancing the delivery of the peptide to that tissuerelative to the amount of underivatized peptide that is delivered to thetissue. In a still further preferred embodiment, the amount ofderivatized peptide delivered to a specific tissue within a selectedtime period is enhanced by derivatization by at least about 20%, morepreferably, at least about 40%, and more preferably still, at leastabout 100%. Presently, preferred biomolecules for targeting applicatio90ns include antibodies, hormones and ligands for cell-surface receptors.

In still a further exemplary embodiment, there is provided as conjugatewith biotin. Thus, for example, a selectively biotinylated peptide iselaborated by the attachment of an avidin or streptavidin moiety bearingone or more modifying groups.

Therapeutic Moieties

In another preferred embodiment, the modified sugar includes atherapeutic moiety. Those of skill in the art will appreciate that thereis overlap between the category of therapeutic moieties andbiomolecules; many biomolecules have therapeutic properties orpotential.

The therapeutic moieties can be agents already accepted for clinical useor they can be drugs whose use is experimental, or whose activity ormechanism of action is under investigation. The therapeutic moieties canhave a proven action in a given disease state or can be onlyhypothesized to show desirable action in a given disease state. In apreferred embodiment, the therapeutic moieties are compounds, which arebeing screened for their ability to interact with a tissue of choice.Therapeutic moieties, which are useful in practicing the instantinvention include drugs from a broad range of drug classes having avariety of pharmacological activities. Preferred therapeutic moietiesare essentially non-fluorescent, or emit such a minimal amount offluorescence that they are inappropriate for use as a fluorescent markerin an assay. Moreover, it is generally preferred to use therapeuticmoieties that are not sugars. An exception to this preference is the useof a sugar that is modified by covalent attachment of another entity,such as a PEG, biomolecule, therapeutic moiety, diagnostic moiety andthe like. In another exemplary embodiment, a therapeutic sugar moiety isconjugated to a linker arm and the sugar-linker arm cassette issubsequently conjugated to a peptide via a method of the invention.

Methods of conjugating therapeutic and diagnostic agents to variousother species are well known to those of skill in the art. See, forexample Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego,1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS,ACS Symposium Series Vol. 469, American Chemical Society, Washington,D.C. 1991.

In an exemplary embodiment, the therapeutic moiety is attached to themodified sugar via a linkage that is cleaved under selected conditions.Exemplary conditions include, but are not limited to, a selected pH(e.g., stomach, intestine, endocytotic vacuole), the presence of anactive enzyme (e.g, esterase, reductase, oxidase), light, heat and thelike. Many cleavable groups are known in the art. See, for example, Junget al., Biochem. Biophys. Acta, 761: 152-162 (1983); Joshi et al., J.Biol. Chem., 265: 14518-14525 (1990); Zarling et al., J. Immunol., 124:913-920 (1980); Bouizar et al., Eur. J. Biochem., 155: 141-147 (1986);Park et al., J. Biol. Chem., 261: 205-210 (1986); Browning et al., J.Immunol., 143: 1859-1867 (1989).

Preparation of Modified Sugars

In general, the sugar moiety and the modifying group are linked togetherthrough the use of reactive groups, which are typically transformed bythe linking process into a new organic functional group or unreactivespecies. The sugar reactive functional group(s), is located at anyposition on the sugar moiety. Reactive groups and classes of reactionsuseful in practicing the present invention are generally those that arewell known in the art of bioconjugate chemistry. Currently favoredclasses of reactions available with reactive sugar moieties are those,which proceed under relatively mild conditions. These include, but arenot limited to nucleophilic substitutions (e.g., reactions of amines andalcohols with acyl halides, active esters), electrophilic substitutions(e.g., enamine reactions) and additions to carbon-carbon andcarbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alderaddition). These and other useful reactions are discussed in, forexample, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons,New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, SanDiego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances inChemistry Series, Vol. 198, American Chemical Society, Washington, D.C.,1982.

Useful reactive functional groups pendent from a sugar nucleus ormodifying group include, but are not limited to:

-   -   (a) carboxyl groups and various derivatives thereof including,        but not limited to, N-hydroxysuccinimide esters,        N-hydroxybenztriazole esters, acid halides, acyl imidazoles,        thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and        aromatic esters;    -   (b) hydroxyl groups, which can be converted to, e.g., esters,        ethers, aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be later displaced        with a nucleophilic group such as, for example, an amine, a        carboxylate anion, thiol anion, carbanion, or an alkoxide ion,        thereby resulting in the covalent attachment of a new group at        the functional group of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, such that subsequent        derivatization is possible via formation of carbonyl derivatives        such as, for example, imines, hydrazones, semicarbazones or        oximes, or via such mechanisms as Grignard addition or        alkyllithium addition;    -   (f) sulfonyl halide groups for subsequent reaction with amines,        for example, to form sulfonamides;    -   (g) thiol groups, which can be, for example, converted to        disulfides or reacted with acyl halides;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc; and    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe reactive sugar nucleus or modifying group. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art understandhow to protect a particular functional group such that it does notinterfere with a chosen set of reaction conditions. For examples ofuseful protecting groups, see, for example, Greene et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

In the discussion that follows, a number of specific examples ofmodified sugars that are useful in practicing the present invention areset forth. In the exemplary embodiments, a sialic acid derivative isutilized as the sugar nucleus to which the modifying group is attached.The focus of the discussion on sialic acid derivatives is for clarity ofillustration only and should not be construed to limit the scope of theinvention. Those of skill in the art will appreciate that a variety ofother sugar moieties can be activated and derivatized in a manneranalogous to that set forth using sialic acid as an example. Forexample, numerous methods are available for modifying galactose,glucose, N-acetylgalactosamine and fucose to name a few sugarsubstrates, which are readily modified by art recognized methods. See,for example, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); and Schaferet al., J. Org. Chem. 65: 24 (2000)).

In an exemplary embodiment, the peptide that is modified by a method ofthe invention is a GLP-1 peptide that has had one or more mutationsintroduced according to the methods of the invention. Theoligosaccharide chains of the glycopeptide lacking a sialic acid andcontaining a terminal galactose residue can be glyco-PEG-ylated,glyco-PPG-ylated or otherwise modified with a modified sialic acid.

In Scheme 1, the amino glycoside 1, is treated with the active ester ofa protected amino acid (e.g., glycine) derivative, converting the sugaramine residue into the corresponding protected amino acid amide adduct.The adduct is treated with an aldolase to form α-hydroxy carboxylate 2.Compound 2 is converted to the corresponding CMP derivative by theaction of CMP-SA synthetase, followed by catalytic hydrogenation of theCMP derivative to produce compound 3. The amine introduced via formationof the glycine adduct is utilized as a locus of PEG or PPG attachment byreacting compound 3 with an activated (m-) PEG or (m-) PPG derivative(e.g., PEG-C(O)NHS, PPG-C(O)NHS), producing 4 or 5, respectively.

Table 1 sets forth representative examples of sugar monophosphates thatare derivatized with a PEG or PPG moiety. Certain of the compounds ofTable 1 are prepared by the method of Scheme 1. Other derivatives areprepared by art-recognized methods. See, for example, Keppler et al.,Glycobiology 11: 11R (2001); and Charter et al., Glycobiology 10: 1049(2000)). Other amine reactive PEG and PPG analogues are commerciallyavailable, or they can be prepared by methods readily accessible tothose of skill in the art.

TABLE 1

The modified sugar phosphates of use in practicing the present inventioncan be substituted in other positions as well as those set forth above.Presently preferred substitutions of sialic acid are set forth inFormula I:

in which X is a linking group, which is preferably selected from —O—,—N(H)—, —S, CH₂—, and —N(R)₂, in which each R is a member independentlyselected from R¹-R⁵. The symbols Y, Z, A and B each represent a groupthat is selected from the group set forth above for the identity of X.X, Y, Z, A and B are each independently selected and, therefore, theycan be the same or different. The symbols R¹, R², R³, R⁴ and R⁵represent H, a water-soluble polymer, therapeutic moiety, biomolecule orother moiety. Alternatively, these symbols represent a linker that isbound to a water-soluble polymer, therapeutic moiety, biomolecule orother moiety.

Exemplary moieties attached to the conjugates disclosed herein include,but are not limited to, PEG derivatives (e.g., alkyl-PEG, acyl-PEG,acyl-alkyl-PEG, alkyl-acyl-PEG carbamoyl-PEG, aryl-PEG), PPG derivatives(e.g., alkyl-PPG, acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-PPGcarbamoyl-PPG, aryl-PPG), therapeutic moieties, diagnostic moieties,mannose-6-phosphate, heparin, heparan, SLe_(x), mannose,mannose-6-phosphate, Sialyl Lewis X, FGF, VFGF, proteins, chondroitin,keratan, dermatan, albumin, integrins, antennary oligosaccharides,peptides and the like. Methods of conjugating the various modifyinggroups to a saccharide moiety are readily accessible to those of skillin the art (POLY (ETHYLENE GLYCOL CHEMISTRY: BIOTECHNICAL AND BIOMEDICALAPPLICATIONS, J. Milton Harris, Ed., Plenum Pub. Corp., 1992; POLY(ETHYLENE GLYCOL) CHEMICAL AND BIOLOGICAL APPLICATIONS, J. MiltonHarris, Ed., ACS Symposium Series No. 680, American Chemical Society,1997; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego,1996; and Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS,ACS Symposium Series Vol. 469, American Chemical Society, Washington,D.C. 1991).

Cross-linking Groups

Preparation of the Modified Sugar for Use in the Methods of the PresentInvention includes attachment of a modifying group to a sugar residueand forming a stable adduct, which is a substrate for aglycosyltransferase. The sugar and modifying group can be coupled by azero- or higher-order cross-linking agent. Exemplary bifunctionalcompounds which can be used for attaching modifying groups tocarbohydrate moieties include, but are not limited to, bifunctionalpoly(ethyleneglycols), polyamides, polyethers, polyesters and the like.General approaches for linking carbohydrates to other molecules areknown in the literature. See, for example, Lee et al., Biochemistry 28:1856 (1989); Bhatia et al., Anal. Biochem. 178: 408 (1989); Janda etal., J. Am. Chem. Soc. 112: 8886 (1990) and Bednarski et al., WO92/18135. In the discussion that follows, the reactive groups aretreated as benign on the sugar moiety of the nascent modified sugar. Thefocus of the discussion is for clarity of illustration. Those of skillin the art will appreciate that the discussion is relevant to reactivegroups on the modifying group as well.

An exemplary strategy involves incorporation of a protected sulfhydrylonto the sugar using the heterobifunctional crosslinker SPDP(n-succinimidyl-3-(2-pyridyldithio)propionate and then deprotecting thesulfhydryl for formation of a disulfide bond with another sulfhydryl onthe modifying group.

If SPDP detrimentally affects the ability of the modified sugar to actas a glycosyltransferase substrate, one of an array of othercrosslinkers such as 2-iminothiolane or N-succinimidylS-acetylthioacetate (SATA) is used to form a disulfide bond.2-iminothiolane reacts with primary amines, instantly incorporating anunprotected sulfhydryl onto the amine-containing molecule. SATA alsoreacts with primary amines, but incorporates a protected sulfhydryl,which is later deacetaylated using hydroxylamine to produce a freesulfhydryl. In each case, the incorporated sulfhydryl is free to reactwith other sulfhydryls or protected sulfhydryl, like SPDP, forming therequired disulfide bond.

The above-described strategy is exemplary, and not limiting, of linkersof use in the invention. Other crosslinkers are available that can beused in different strategies for crosslinking the modifying group to thepeptide. For example, TPCH(S-(2-thiopyridyl)-L-cysteine hydrazide andTPMPH ((S-(2-thiopyridyl) mercapto-propionohydrazide) react withcarbohydrate moieties that have been previously oxidized by mildperiodate treatment, thus forming a hydrazone bond between the hydrazideportion of the crosslinker and the periodate generated aldehydes. TPCHand TPMPH introduce a 2-pyridylthione protected sulfhydryl group ontothe sugar, which can be deprotected with DTT and then subsequently usedfor conjugation, such as forming disulfide bonds between components.

If disulfide bonding is found unsuitable for producing stable modifiedsugars, other crosslinkers may be used that incorporate more stablebonds between components. The heterobifunctional crosslinkers GMBS(N-gama-malimidobutyryloxy)succinimide) and SMCC (succinimidyl4-(N-maleimido-methyl)cyclohexane) react with primary amines, thusintroducing a maleimide group onto the component. The maleimide groupcan subsequently react with sulfhydryls on the other component, whichcan be introduced by previously mentioned crosslinkers, thus forming astable thioether bond between the components. If steric hindrancebetween components interferes with either component's activity or theability of the modified sugar to act as a glycosyltransferase substrate,crosslinkers can be used which introduce long spacer arms betweencomponents and include derivatives of some of the previously mentionedcrosslinkers (i.e., SPDP). Thus, there is an abundance of suitablecrosslinkers, which are useful; each of which is selected depending onthe effects it has on optimal peptide conjugate and modified sugarproduction.

A variety of reagents are used to modify the components of the modifiedsugar with intramolecular chemical crosslinks (for reviews ofcrosslinking reagents and crosslinking procedures see: Wold, F., Meth.Enzymol. 25: 623-651, 1972; Weetall, H. H., and Cooney, D. A., In:ENZYMES AS DRUGS. (Holcenberg, and Roberts, eds.) pp. 395-442, Wiley,New York, 1981; Ji, T. H., Meth. Enzymol. 91: 580-609, 1983; Mattson etal., Mol. Biol. Rep. 17: 167-183, 1993, all of which are incorporatedherein by reference). Preferred crosslinking reagents are derived fromvarious zero-length, homo-bifunctional, and hetero-bifunctionalcrosslinking reagents. Zero-length crosslinking reagents include directconjugation of two intrinsic chemical groups with no introduction ofextrinsic material. Agents that catalyze formation of a disulfide bondbelong to this category. Another example is reagents that inducecondensation of a carboxyl and a primary amino group to form an amidebond such as carbodiimides, ethylchloroformate, Woodward's reagent K(2-ethyl-5-phenylisoxazolium-3′-sulfonate), and carbonyldiimidazole. Inaddition to these chemical reagents, the enzyme transglutaminase(glutamyl-peptide γ-glutamyltransferase; EC 2.3.2.13) may be used aszero-length crosslinking reagent. This enzyme catalyzes acyl transferreactions at carboxamide groups of protein-bound glutaminyl residues,usually with a primary amino group as substrate. Preferred homo- andhetero-bifunctional reagents contain two identical or two dissimilarsites, respectively, which may be reactive for amino, sulfhydryl,guanidino, indole, or nonspecific groups.

i. Preferred Specific Sites in Crosslinking Reagents

1. Amino-Reactive Groups

In one preferred embodiment, the sites on the cross-linker areamino-reactive groups. Useful non-limiting examples of amino-reactivegroups include N-hydroxysuccinimide (NHS) esters, imidoesters,isocyanates, acylhalides, arylazides, p-nitrophenyl esters, aldehydes,and sulfonyl chlorides.

NHS esters react preferentially with the primary (including aromatic)amino groups of a modified sugar component. The imidazole groups ofhistidines are known to compete with primary amines for reaction, butthe reaction products are unstable and readily hydrolyzed. The reactioninvolves the nucleophilic attack of an amine on the acid carboxyl of anNHS ester to form an amide, releasing the N-hydroxysuccinimide. Thus,the positive charge of the original amino group is lost.

Imidoesters are the most specific acylating reagents for reaction withthe amine groups of the modified sugar components. At a pH between 7 and10, imidoesters react only with primary amines. Primary amines attackimidates nucleophilically to produce an intermediate that breaks down toamidine at high pH or to a new imidate at low pH. The new imidate canreact with another primary amine, thus crosslinking two amino groups, acase of a putatively monofunctional imidate reacting bifunctionally. Theprincipal product of reaction with primary amines is an amidine that isa stronger base than the original amine. The positive charge of theoriginal amino group is therefore retained.

Isocyanates (and isothiocyanates) react with the primary amines of themodified sugar components to form stable bonds. Their reactions withsulfhydryl, imidazole, and tyrosyl groups give relatively unstableproducts.

Acylazides are also used as amino-specific reagents in whichnucleophilic amines of the affinity component attack acidic carboxylgroups under slightly alkaline conditions, e.g. pH 8.5.

Arylhalides such as 1,5-difluoro-2,4-dinitrobenzene react preferentiallywith the amino groups and tyrosine phenolic groups of modified sugarcomponents, but also with sulfhydryl and imidazole groups.

p-Nitrophenyl esters of mono- and dicarboxylic acids are also usefulamino-reactive groups. Although the reagent specificity is not veryhigh, α- and ε-amino groups appear to react most rapidly.

Aldehydes such as glutaraldehyde react with primary amines of modifiedsugar. Although unstable Schiff bases are formed upon reaction of theamino groups with the aldehydes of the aldehydes, glutaraldehyde iscapable of modifying the modified sugar with stable crosslinks. At pH6-8, the pH of typical crosslinking conditions, the cyclic polymersundergo a dehydration to form α-β unsaturated aldehyde polymers. Schiffbases, however, are stable, when conjugated to another double bond. Theresonant interaction of both double bonds prevents hydrolysis of theSchiff linkage. Furthermore, amines at high local concentrations canattack the ethylenic double bond to form a stable Michael additionproduct.

Aromatic sulfonyl chlorides react with a variety of sites of themodified sugar components, but reaction with the amino groups is themost important, resulting in a stable sulfonamide linkage.

2. Sulfhydryl-Reactive Groups

In another preferred embodiment, the sites are sulfhydryl-reactivegroups. Useful, non-limiting examples of sulfhydryl-reactive groupsinclude maleimides, alkyl halides, pyridyl disulfides, andthiophthalimides.

Maleimides react preferentially with the sulfhydryl group of themodified sugar components to form stable thioether bonds. They alsoreact at a much slower rate with primary amino groups and the imidazolegroups of histidines. However, at pH 7 the maleimide group can beconsidered a sulfhydryl-specific group, since at this pH the reactionrate of simple thiols is 1000-fold greater than that of thecorresponding amine.

Alkyl halides react with sulfhydryl groups, sulfides, imidazoles, andamino groups. At neutral to slightly alkaline pH, however, alkyl halidesreact primarily with sulfhydryl groups to form stable thioether bonds.At higher pH, reaction with amino groups is favored.

Pyridyl disulfides react with free sulfhydryls via disulfide exchange togive mixed disulfides. As a result, pyridyl disulfides are the mostspecific sulfhydryl-reactive groups.

Thiophthalimides react with free sulfhydryl groups to form disulfides.

3. Carboxyl-Reactive Residue

In another embodiment, carbodiimides soluble in both water and organicsolvent, are used as carboxyl-reactive reagents. These compounds reactwith free carboxyl groups forming a pseudourea that can then couple toavailable amines yielding an amide linkage teach how to modify acarboxyl group with carbodiimde (Yamada et al., Biochemistry 20:4836-4842, 1981).

ii. Preferred Nonspecific Sites in Crosslinking Reagents

In addition to the use of site-specific reactive moieties, the presentinvention contemplates the use of non-specific reactive groups to linkthe sugar to the modifying group.

Exemplary non-specific cross-linkers include photoactivatable groups,completely inert in the dark, which are converted to reactive speciesupon absorption of a photon of appropriate energy. In one preferredembodiment, photoactivatable groups are selected from precursors ofnitrenes generated upon heating or photolysis of azides.Electron-deficient nitrenes are extremely reactive and can react with avariety of chemical bonds including N—H, O—H, C—H, and C═C. Althoughthree types of azides (aryl, alkyl, and acyl derivatives) may beemployed, arylazides are presently preferred. The reactivity ofarylazides upon photolysis is better with N—H and O—H than C—H bonds.Electron-deficient arylnitrenes rapidly ring-expand to formdehydroazepines, which tend to react with nucleophiles, rather than formC—H insertion products. The reactivity of arylazides can be increased bythe presence of electron-withdrawing substituents such as nitro orhydroxyl groups in the ring. Such substituents push the absorptionmaximum of arylazides to longer wavelength. Unsubstituted arylazideshave an absorption maximum in the range of 260-280 nm, while hydroxy andnitroarylazides absorb significant light beyond 305 nm. Therefore,hydroxy and nitroarylazides are most preferable since they allow toemploy less harmful photolysis conditions for the affinity componentthan unsubstituted arylazides.

In another preferred embodiment, photoactivatable groups are selectedfrom fluorinated arylazides. The photolysis products of fluorinatedarylazides are arylnitrenes, all of which undergo the characteristicreactions of this group, including C—H bond insertion, with highefficiency (Keana et al., J. Org. Chem. 55: 3640-3647, 1990).

In another embodiment, photoactivatable groups are selected frombenzophenone residues. Benzophenone reagents generally give highercrosslinking yields than arylazide reagents.

In another embodiment, photoactivatable groups are selected from diazocompounds, which form an electron-deficient carbene upon photolysis.These carbenes undergo a variety of reactions including insertion intoC—H bonds, addition to double bonds (including aromatic systems),hydrogen attraction and coordination to nucleophilic centers to givecarbon ions.

In still another embodiment, photoactivatable groups are selected fromdiazopyruvates. For example, the p-nitrophenyl ester of p-nitrophenyldiazopyruvate reacts with aliphatic amines to give diazopyruvic acidamides that undergo ultraviolet photolysis to form aldehydes. Thephotolyzed diazopyruvate-modified affinity component will react likeformaldehyde or glutaraldehyde forming crosslinks.

iii. Homobifunctional Reagents1. Homobifunctional Crosslinkers Reactive with Primary Amines

Synthesis, properties, and applications of amine-reactive cross-linkersare commercially described in the literature (for reviews ofcrosslinking procedures and reagents, see above). Many reagents areavailable (e.g., Pierce Chemical Company, Rockford, Ill.; Sigma ChemicalCompany, St. Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

Preferred, non-limiting examples of homobifunctional NHS esters includedisuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartarate (DST),disulfosuccinimidyl tartarate (sulfo-DST),bis-2-(succinimidooxycarbonyloxy)ethylsulfone (BSOCOES),bis-2-(sulfosuccinimidooxy-carbonyloxy)ethylsulfone (sulfo-BSOCOES),ethylene glycolbis(succinimidylsuccinate) (EGS), ethyleneglycolbis(sulfosuccinimidylsuccinate) (sulfo-EGS),dithiobis(succinimidyl-propionate (DSP), anddithiobis(sulfosuccinimidylpropionate (sulfo-DSP). Preferred,non-limiting examples of homobifunctional imidoesters include dimethylmalonimidate (DMM), dimethyl succinimidate (DMSC), dimethyl adipimidate(DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-oxydipropionimidate (DODP),dimethyl-3,3′-(methylenedioxy)dipropionimidate (DMDP),dimethyl-,3′-(dimethylenedioxy)dipropionimidate (DDDP),dimethyl-3,3′-(tetramethylenedioxy)-dipropionimidate (DTDP), anddimethyl-3,3′-dithiobispropionimidate (DTBP).

Preferred, non-limiting examples of homobifunctional isothiocyanatesinclude: p-phenylenediisothiocyanate (DITC), and4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS).

Preferred, non-limiting examples of homobifunctional isocyanates includexylene-diisocyanate, toluene-2,4-diisocyanate,toluene-2-isocyanate-4-isothiocyanate,3-methoxydiphenylmethane-4,4′-diisocyanate,2,2′-dicarboxy-4,4′-azophenyldiisocyanate, andhexamethylenediisocyanate.

Preferred, non-limiting examples of homobifunctional arylhalides include1,5-difluoro-2,4-dinitrobenzene (DFDNB), and4,4′-difluoro-3,3′-dinitrophenyl-sulfone.

Preferred, non-limiting examples of homobifunctional aliphatic aldehydereagents include glyoxal, malondialdehyde, and glutaraldehyde.

Preferred, non-limiting examples of homobifunctional acylating reagentsinclude nitrophenyl esters of dicarboxylic acids.

Preferred, non-limiting examples of homobifunctional aromatic sulfonylchlorides include phenol-2,4-disulfonyl chloride, andα-naphthol-2,4-disulfonyl chloride.

Preferred, non-limiting examples of additional amino-reactivehomobifunctional reagents include erythritolbiscarbonate which reactswith amines to give biscarbamates.

2. Homobifunctional Crosslinkers Reactive with Free Sulfhydryl Groups

Synthesis, properties, and applications of such reagents are describedin the literature (for reviews of crosslinking procedures and reagents,see above). Many of the reagents are commercially available (e.g.,Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

Preferred, non-limiting examples of homobifunctional maleimides includebismaleimidohexane (BMH), N,N′-(1,3-phenylene) bismaleimide,N,N′-(1,2-phenylene)bismaleimide, azophenyldimaleimide, andbis(N-maleimidomethyl)ether.

Preferred, non-limiting examples of homobifunctional pyridyl disulfidesinclude 1,4-di-3′-(2′-pyridyldithio)propionamidobutane (DPDPB).

Preferred, non-limiting examples of homobifunctional alkyl halidesinclude 2,2′-dicarboxy-4,4′-diiodoacetamidoazobenzene,α,α′-diiodo-p-xylenesulfonic acid, α,α′-dibromo-p-xylenesulfonic acid,N,N′-bis(b-bromoethyl)benzylamine, N,N′-di(bromoacetyl)phenylthydrazine,and 1,2-di(bromoacetyl)amino-3-phenylpropane.

3. Homobifunctional Photoactivatable Crosslinkers

Synthesis, properties, and applications of such reagents are describedin the literature (for reviews of crosslinking procedures and reagents,see above). Some of the reagents are commercially available (e.g.,Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

Preferred, non-limiting examples of homobifunctional photoactivatablecrosslinker include bis-β-(4-azidosalicylamido)ethyldisulfide (BASED),di-N-(2-nitro-4-azidophenyl)-cystamine-S,S-dioxide (DNCO), and4,4′-dithiobisphenylazide.

iv. HeteroBifunctional Reagents1. Amino-Reactive HeteroBifunctional Reagents with a Pyridyl DisulfideMoiety

Synthesis, properties, and applications of such reagents are describedin the literature (for reviews of crosslinking procedures and reagents,see above). Many of the reagents are commercially available (e.g.,Pierce Chemical Company, Rockford, Ill.; Sigma Chemical Company, St.Louis, Mo.; Molecular Probes, Inc., Eugene, Oreg.).

Preferred, non-limiting examples of hetero-bifunctional reagents with apyridyl disulfide moiety and an amino-reactive NHS ester includeN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl6-3-(2-pyridyldithio)propionamidohexanoate (LC-SPDP), sulfosuccinimidyl6-3-(2-pyridyldithio)propionamidohexanoate (sulfo-LCSPDP),4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT),and sulfosuccinimidyl 6-α-methyl-α-(2-pyridyldithio)toluamidohexanoate(sulfo-LC-SMPT).

2. Amino-Reactive HeteroBifunctional Reagents with a Maleimide Moiety

Synthesis, properties, and applications of such reagents are describedin the literature. Preferred, non-limiting examples ofhetero-bifunctional reagents with a maleimide moiety and anamino-reactive NHS ester include succinimidyl maleimidylacetate (AMAS),succinimidyl 3-maleimidylpropionate (BMPS),N-γ-maleimidobutyryloxysuccinimide ester(GMBS)N-γ-maleimidobutyryloxysulfo succinimide ester (sulfo-GMBS)succinimidyl 6-maleimidylhexanoate (EMCS), succinimidyl3-maleimidylbenzoate (SMB), m-maleimidobenzoyl-N-hydroxysuccinimideester (MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester(sulfo-MBS), succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), andsulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).

3. Amino-Reactive HeteroBifunctional Reagents with an Alkyl HalideMoiety

Synthesis, properties, and applications of such reagents are describedin the literature Preferred, non-limiting examples ofhetero-bifunctional reagents with an alkyl halide moiety and anamino-reactive NHS ester includeN-succinimidyl-(4-iodoacetyl)aminobenzoate (SIAB),sulfosuccinimidyl-(4-iodoacetyl)aminobenzoate (sulfo-SIAB),succinimidyl-6-(iodoacetyl)aminohexanoate (SIAX),succinimidyl-6-(6-((iodoacetyl)-amino)hexanoylamino)hexanoate (SIAXX),succinimidyl-6-(((4-(iodoacetyl)-amino)-methyl)-cyclohexane-1-carbonyl)aminohexanoate(SIACX), andsuccinimidyl-4((iodoacetyl)-amino)methylcyclohexane-1-carboxylate(SIAC).

A preferred example of a hetero-bifunctional reagent with anamino-reactive NHS ester and an alkyl dihalide moiety isN-hydroxysuccinimidyl 2,3-dibromopropionate (SDBP). SDBP introducesintramolecular crosslinks to the affinity component by conjugating itsamino groups. The reactivity of the dibromopropionyl moiety towardsprimary amine groups is controlled by the reaction temperature (McKenzieet al., Protein Chem. 7: 581-592 (1988)).

Preferred, non-limiting examples of hetero-bifunctional reagents with analkyl halide moiety and an amino-reactive p-nitrophenyl ester moietyinclude p-nitrophenyl iodoacetate (NPIA).

Other cross-linking agents are known to those of skill in the art. See,for example, Pomato et al., U.S. Pat. No. 5,965,106. It is within theabilities of one of skill in the art to choose an appropriatecross-linking agent for a particular application.

v. Cleavable Linker Groups

In yet a further embodiment, the linker group is provided with a groupthat can be cleaved to release the modifying group from the sugarresidue. Many cleaveable groups are known in the art. See, for example,Jung et al., Biochem. Biophys. Acta 761: 152-162 (1983); Joshi et al.,J. Biol. Chem. 265: 14518-14525 (1990); Zarling et al., J. Immunol. 124:913-920 (1980); Bouizar et al., Eur. J. Biochem. 155: 141-147 (1986);Park et al., J. Biol. Chem. 261: 205-210 (1986); Browning et al., J.Immunol. 143: 1859-1867 (1989). Moreover a broad range of cleavable,bifunctional (both homo- and hetero-bifunctional) linker groups iscommercially available from suppliers such as Pierce.

Exemplary cleaveable moieties can be cleaved using light, heat orreagents such as thiols, hydroxylamine, bases, periodate and the like.Moreover, certain preferred groups are cleaved in vivo in response tobeing endocytized (e.g., cis-aconityl; see, Shen et al., Biochem.Biophys. Res. Commun. 102: 1048 (1991)). Preferred cleaveable groupscomprise a cleaveable moiety which is a member selected from the groupconsisting of disulfide, ester, imide, carbonate, nitrobenzyl, phenacyland benzoin groups.

Conjugation of Modified Sugars to Peptides

The modified sugars are conjugated to a glycosylated or non-glycosylatedpeptide using an appropriate enzyme to mediate the conjugation.Preferably, the concentrations of the modified donor sugar(s), enzyme(s)and acceptor peptide(s) are selected such that glycosylation proceedsuntil the acceptor is consumed. The considerations discussed below,while set forth in the context of a sialyltransferase, are generallyapplicable to other glycosyltransferase reactions.

A number of methods of using glycosyltransferases to synthesize desiredoligosaccharide structures are known and are generally applicable to theinstant invention. Exemplary methods are described, for instance, WO96/32491, Ito et al., Pure Appl. Chem. 65: 753 (1993), and U.S. Pat.Nos. 5,352,670, 5,374,541, and 5,545,553.

The present invention is practiced using a single glycosyltransferase ora combination of glycosyltransferases. For example, one can use acombination of a sialyltransferase and a galactosyltransferase. In thoseembodiments using more than one enzyme, the enzymes and substrates arepreferably combined in an initial reaction mixture, or the enzymes andreagents for a second enzymatic reaction are added to the reactionmedium once the first enzymatic reaction is complete or nearly complete.By conducting two enzymatic reactions in sequence in a single vessel,overall yields are improved over procedures in which an intermediatespecies is isolated. Moreover, cleanup and disposal of extra solventsand by-products is reduced.

In a preferred embodiment, each of the first and second enzyme is aglycosyltransferase. In another preferred embodiment, one enzyme is anendoglycosidase. In an additional preferred embodiment, more than twoenzymes are used to assemble the modified glycoprotein of the invention.The enzymes are used to alter a saccharide structure on the peptide atany point either before or after the addition of the modified sugar tothe peptide.

The O-linked glycosyl moieties of the conjugates of the invention aregenerally originate with a GalNAc moiety that is attached to thepeptide. Any member of the family of GalNAc transferases can be used tobind a GalNAc moiety to the peptide (Hassan H, Bennett E P, Mandel U,Hollingsworth M A, and Clausen H (2000). Control of Mucin-TypeO-Glycosylation: O-Glycan Occupancy is Directed by SubstrateSpecificities of Polypeptide GalNAc-Transferases. (Eds. Ernst, Hart, andSinay). Wiley-VCH chapter “Carbohydrates in Chemistry and Biology—aComprehensive Handbook”, 273-292). The GalNAc moiety itself can be theintact glycosyl linker. Alternatively, the saccharyl residue is builtout using one more enzyme and one or more appropriate glycosyl substratefor the enzyme, the modified sugar being added to the built out glycosylmoiety.

In another embodiment, the method makes use of one or more exo- orendoglycosidase. The glycosidase is typically a mutant, which isengineered to form glycosyl bonds rather than cleave them. The mutantglycanase typically includes a substitution of an amino acid residue foran active site acidic amino acid residue. For example, when theendoglycanase is endo-H, the substituted active site residues willtypically be Asp at position 130, Glu at position 132 or a combinationthereof. The amino acids are generally replaced with serine, alanine,asparagine, or glutamine.

The mutant enzyme catalyzes the reaction, usually by a synthesis stepthat is analogous to the reverse reaction of the endoglycanasehydrolysis step. In these embodiments, the glycosyl donor molecule(e.g., a desired oligo- or mono-saccharide structure) contains a leavinggroup and the reaction proceeds with the addition of the donor moleculeto a GlcNAc residue on the protein. For example, the leaving group canbe a halogen, such as fluoride. In other embodiments, the leaving groupis a Asn, or a Asn-peptide moiety. In yet further embodiments, theGlcNAc residue on the glycosyl donor molecule is modified. For example,the GlcNAc residue may comprise a 1,2 oxazoline moiety.

In a preferred embodiment, each of the enzymes utilized to produce aconjugate of the invention are present in a catalytic amount. Thecatalytic amount of a particular enzyme varies according to theconcentration of that enzyme's substrate as well as to reactionconditions such as temperature, time and pH value. Means for determiningthe catalytic amount for a given enzyme under preselected substrateconcentrations and reaction conditions are well known to those of skillin the art.

The temperature at which an above process is carried out can range fromjust above freezing to the temperature at which the most sensitiveenzyme denatures. Preferred temperature ranges are about 0° C. to about55° C., and more preferably about 20° C. to about 30° C. In anotherexemplary embodiment, one or more components of the present method areconducted at an elevated temperature using a thermophilic enzyme.

The reaction mixture is maintained for a period of time sufficient forthe acceptor to be glycosylated, thereby forming the desired conjugate.Some of the conjugate can often be detected after a few hours, withrecoverable amounts usually being obtained within 24 hours or less.Those of skill in the art understand that the rate of reaction isdependent on a number of variable factors (e.g, enzyme concentration,donor concentration, acceptor concentration, temperature, solventvolume), which are optimized for a selected system.

The present invention also provides for the industrial-scale productionof modified peptides. As used herein, an industrial scale generallyproduces at least about 250 mg, preferably at least about 500 mg, andmore preferably at least about 1 gram of finished, purified conjugate,preferably after a single reaction cycle, i.e., the conjugate is not acombination the reaction products from identical, consecutively iteratedsynthesis cycles.

In the discussion that follows, the invention is exemplified by theconjugation of modified sialic acid moieties to a glycosylated peptide.The exemplary modified sialic acid is labeled with (m-) PEG. The focusof the following discussion on the use of PEG-modified sialic acid andglycosylated peptides is for clarity of illustration and is not intendedto imply that the invention is limited to the conjugation of these twopartners. One of skill understands that the discussion is generallyapplicable to the additions of modified glycosyl moieties other thansialic acid. Moreover, the discussion is equally applicable to themodification of a glycosyl unit with agents other than PEG includingother water-soluble polymers, therapeutic moieties, and biomolecules.

An enzymatic approach can be used for the selective introduction of (m-)PEG-ylated or (m-) PPG-ylated carbohydrates onto a peptide orglycopeptide. The method utilizes modified sugars containing PEG, PPG,or a masked reactive functional group, and is combined with theappropriate glycosyltransferase or glycosynthase. By selecting theglycosyltransferase that will make the desired carbohydrate linkage andutilizing the modified sugar as the donor substrate, the PEG or PPG canbe introduced directly onto the peptide backbone, onto existing sugarresidues of a glycopeptide or onto sugar residues that have been addedto a peptide.

An acceptor for the sialyltransferase is present on the peptide to bemodified by the methods of the present invention either as a naturallyoccurring structure or one placed there recombinantly, enzymatically orchemically. Suitable acceptors, include, for example, galactosylacceptors such as GalNAc, Galβ1,4GlcNAc, Galβ1,4GalNAc, Galβ1,3GalNAc,lacto-N-tetraose, Galβ1,3GlcNAc, Galβ1,3Ara, Galβ1,6GlcNAc, Galβ1,4Glc(lactose), and other acceptors known to those of skill in the art (see,e.g., Paulson et al., J. Biol. Chem. 253: 5617-5624 (1978)).

In one embodiment, an acceptor for the sialyltransferase is present onthe glycopeptide to be modified upon in vivo synthesis of theglycopeptide. Such glycopeptides can be sialylated using the claimedmethods without prior modification of the glycosylation pattern of theglycopeptide. Alternatively, the methods of the invention can be used tosialylate a peptide that does not include a suitable acceptor; one firstmodifies the peptide to include an acceptor by methods known to those ofskill in the art. In an exemplary embodiment, a GalNAc residue is addedto an O-linked glycosylation site by the action of a GalNAc transferase.Hassan H, Bennett E P, Mandel U, Hollingsworth M A, and Clausen H(2000). Control of Mucin-Type O-Glycosylation: O-Glycan Occupancy isDirected by Substrate Specificities of Polypeptide GalNAc-Transferases.(Eds. Ernst, Hart, and Sinay). Wiley-VCH chapter “Carbohydrates inChemistry and Biology—a Comprehension Handbook”, 273-292.

In an exemplary embodiment, the galactosyl acceptor is assembled byattaching a galactose residue to an appropriate acceptor linked to thepeptide, e.g., a GalNAc. The method includes incubating the peptide tobe modified with a reaction mixture that contains a suitable amount of agalactosyltransferase (e.g., Galβ1,3 or Galβ1,4), and a suitablegalactosyl donor (e.g., UDP-galactose). The reaction is allowed toproceed substantially to completion or, alternatively, the reaction isterminated when a preselected amount of the galactose residue is added.Other methods of assembling a selected saccharide acceptor will beapparent to those of skill in the art.

In yet another embodiment, glycopeptide-linked oligosaccharides arefirst “trimmed,” either in whole or in part, to expose either anacceptor for the sialyltransferase or a moiety to which one or moreappropriate residues can be added to obtain a suitable acceptor. Enzymessuch as glycosyltransferases and endoglycosidases (see, for example U.S.Pat. No. 5,716,812) are useful for the attaching and trimming reactions.

In the discussion that follows, the method of the invention isexemplified by the use of modified sugars having a water-soluble polymerattached thereto. The focus of the discussion is for clarity ofillustration. Those of skill will appreciate that the discussion isequally relevant to those embodiments in which the modified sugar bearsa therapeutic moiety, biomolecule or the like.

In an exemplary embodiment, an O-linked carbohydrate residue is“trimmed” prior to the addition of the modified sugar. For example aGalNAc-Gal residue is trimmed back to GalNAc. A modified sugar bearing awater-soluble polymer is conjugated to one or more of the sugar residuesexposed by the “trimming.” In one example, a glycopeptide is “trimmed”and a water-soluble polymer is added to the resulting O-side chain aminoacid or glycopeptide glycan via a saccharyl moiety, e.g., Sia, Gal orGalNAc moiety conjugated to the water-soluble polymer. The modifiedsaccharyl moiety is attached to an acceptor site on the “trimmed”glycopeptide. Alternatively, an unmodified saccharyl moiety, e.g., Galcan be added the terminus of the O-linked glycan.

In another exemplary embodiment, a water-soluble polymer is added to aGalNAc residue via a modified sugar having a galactose residue.Alternatively, an unmodified Gal can be added to the terminal GalNAcresidue.

In yet a further example, a water-soluble polymer is added onto a Galresidue using a modified sialic acid.

In another exemplary embodiment, an O-linked glycosyl residue is“trimmed back” to the GalNAc attached to the amino acid. In one example,a water-soluble polymer is added via a Gal modified with the polymer.Alternatively, an unmodified Gal is added to the GalNAc, followed by aGal with an attached water-soluble polymer. In yet another embodiment,one or more unmodified Gal residue is added to the GalNAc, followed by asialic acid moiety modified with a water-soluble polymer.

The exemplary embodiments discussed above provide an illustration of thepower of the methods set forth herein. Using the methods of theinvention, it is possible to “trim back” and build up a carbohydrateresidue of substantially any desired structure. The modified sugar canbe added to the termini of the carbohydrate moiety as set forth above,or it can be intermediate between the peptide core and the terminus ofthe carbohydrate.

In an exemplary embodiment, the water-soluble-polymer is added to aterminal Gal residue using a polymer modified sialic acid. Anappropriate sialyltransferase is used to add a modified sialic acid. Theapproach is summarized in Scheme 2.

In yet a further approach, summarized in Scheme 3, a masked reactivefunctionality is present on the sialic acid. The masked reactive groupis preferably unaffected by the conditions used to attach the modifiedsialic acid to the peptide. After the covalent attachment of themodified sialic acid to the peptide, the mask is removed and the peptideis conjugated with an agent such as PEG, PPG, a therapeutic moiety,biomolecule or other agent. The agent is conjugated to the peptide in aspecific manner by its reaction with the unmasked reactive group on themodified sugar residue.

Any modified sugar can be used with its appropriate glycosyltransferase,depending on the terminal sugars of the oligosaccharide side chains ofthe glycopeptide (Table 2). As discussed above, the terminal sugar ofthe glycopeptide required for introduction of the PEG-ylated orPPGylated structure can be introduced naturally during expression or itcan be produced post expression using the appropriate glycosidase(s),glycosyltransferase(s) or mix of glycosidase(s) andglycosyltransferase(s).

TABLE 2

X = O, NH, S, CH₂, N—(R₁₋₅)₂. Y = X; Z = X; A = X; B = X. Q = H₂, O, S,NH, N—R. R, R₁₋₄ = H, Linker-M, M. M = Ligand of interest Ligand ofinterest = acyl-PEG, acyl-PPG, alkyl-PEG, acyl-alkyl-PEG,acyl-alkyl-PEG, carbamoyl-PEG, carbamoyl-PPG, PEG, PPG, acyl-aryl-PEG,acyl-aryl-PPG, aryl-PEG, aryl-PPG, Mannose-₆-phosphate, heparin,heparan, SLex, Mannose, FGF, VFGF, protein, chondroitin, keratan,dermatan, albumin, integrins, peptides, etc.

In an alternative embodiment, the modified sugar is added directly tothe peptide backbone using a glycosyltransferase known to transfer sugarresidues to the O-linked glycosylation site on the peptide backbone.This exemplary embodiment is set forth in Scheme 4. Exemplaryglycosyltransferases useful in practicing the present invention include,but are not limited to, GalNAc transferases (GalNAc T1-20), GlcNActransferases, fucosyltransferases, glucosyltransferases,xylosyltransferases, mannosyltransferases and the like. Use of thisapproach allows the direct addition of modified sugars onto peptidesthat lack any carbohydrates or, alternatively, onto existingglycopeptides. In both cases, the addition of the modified sugar occursat specific positions on the peptide backbone as defined by thesubstrate specificity of the glycosyltransferase and not in a randommanner as occurs during modification of a protein's peptide backboneusing chemical methods. An array of agents can be introduced intoproteins or glycopeptides that lack the glycosyltransferase substratepeptide sequence by engineering the appropriate amino acid sequence intothe polypeptide chain.

In each of the exemplary embodiments set forth above, one or moreadditional chemical or enzymatic modification steps can be utilizedfollowing the conjugation of the modified sugar to the peptide. In anexemplary embodiment, an enzyme (e.g., fucosyltransferase) is used toappend a glycosyl unit (e.g., fucose) onto the terminal modified sugarattached to the peptide. In another example, an enzymatic reaction isutilized to “cap” (e.g., sialylate) sites to which the modified sugarfailed to conjugate. Alternatively, a chemical reaction is utilized toalter the structure of the conjugated modified sugar. For example, theconjugated modified sugar is reacted with agents that stabilize ordestabilize its linkage with the peptide component to which the modifiedsugar is attached. In another example, a component of the modified sugaris deprotected following its conjugation to the peptide. One of skillwill appreciate that there is an array of enzymatic and chemicalprocedures that are useful in the methods of the invention at a stageafter the modified sugar is conjugated to the peptide. Furtherelaboration of the modified sugar-peptide conjugate is within the scopeof the invention.

In another exemplary embodiment, the glycopeptide is conjugated to atargeting agent, e.g., transferrin (to deliver the peptide across theblood-brain barrier, and to endosomes), carnitine (to deliver thepeptide to muscle cells; see, for example, LeBorgne et al., Biochem.Pharmacol. 59: 1357-63 (2000), and phosphonates, e.g., bisphosphonate(to target the peptide to bone and other calciferous tissues; see, forexample, Modern Drug Discovery, August 2002, page 10). Other agentsuseful for targeting are apparent to those of skill in the art. Forexample, glucose, glutamine and IGF are also useful to target muscle.

The targeting moiety and therapeutic peptide are conjugated by anymethod discussed herein or otherwise known in the art. Those of skillwill appreciate that peptides in addition to those set forth above canalso be derivatized as set forth herein. Exemplary peptides are setforth in the Appendix attached to copending, commonly owned U.S.Provisional Patent Application No. 60/328,523 filed Oct. 10, 2001.

In an exemplary embodiment, the targeting agent and the therapeuticpeptide are coupled via a linker moiety. In this embodiment, at leastone of the therapeutic peptide or the targeting agent is coupled to thelinker moiety via an intact glycosyl linking group according to a methodof the invention. In an exemplary embodiment, the linker moiety includesa poly(ether) such as poly(ethylene glycol). In another exemplaryembodiment, the linker moiety includes at least one bond that isdegraded in vivo, releasing the therapeutic peptide from the targetingagent, following delivery of the conjugate to the targeted tissue orregion of the body.

In yet another exemplary embodiment, the in vivo distribution of thetherapeutic moiety is altered via altering a glycoform on thetherapeutic moiety without conjugating the therapeutic peptide to atargeting moiety. For example, the therapeutic peptide can be shuntedaway from uptake by the reticuloendothelial system by capping a terminalgalactose moiety of a glycosyl group with sialic acid (or a derivativethereof).

i. Enzymes

1. Glycosyltransferases

Glycosyltransferases catalyze the addition of activated sugars (donorNDP-sugars), in a step-wise fashion, to a protein, glycopeptide, lipidor glycolipid or to the non-reducing end of a growing oligosaccharide.N-linked glycopeptides are synthesized via a transferase and alipid-linked oligosaccharide donor Dol-PP-NAG₂Glc₃Man₉ in an en blocktransfer followed by trimming of the core. In this case the nature ofthe “core” saccharide is somewhat different from subsequent attachments.A very large number of glycosyltransferases are known in the art.

The glycosyltransferase to be used in the present invention may be anyas long as it can utilize the modified sugar as a sugar donor. Examplesof such enzymes include Leloir pathway glycosyltransferase, such asgalactosyltransferase, N-acetylglucosaminyltransferase,N-acetylgalactosaminyltransferase, fucosyltransferase,sialyltransferase, mannosyltransferase, xylosyltransferase,glucurononyltransferase and the like.

For enzymatic saccharide syntheses that involve glycosyltransferasereactions, glycosyltransferase can be cloned, or isolated from anysource. Many cloned glycosyltransferases are known, as are theirpolynucleotide sequences. See, e.g., “The WWW Guide To ClonedGlycosyltransferases,” (http://www.vei.co.uk/TGN/gt_guide.htm).Glycosyltransferase amino acid sequences and nucleotide sequencesencoding glycosyltransferases from which the amino acid sequences can bededuced are also found in various publicly available databases,including GenBank, Swiss-Prot, EMBL, and others.

Glycosyltransferases that can be employed in the methods of theinvention include, but are not limited to, galactosyltransferases,fucosyltransferases, glucosyltransferases,N-acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases,glucuronyltransferases, sialyltransferases, mannosyltransferases,glucuronic acid transferases, galacturonic acid transferases, andoligosaccharyltransferases. Suitable glycosyltransferases include thoseobtained from eukaryotes, as well as from prokaryotes.

DNA encoding glycosyltransferases may be obtained by chemical synthesis,by screening reverse transcripts of mRNA from appropriate cells or cellline cultures, by screening genomic libraries from appropriate cells, orby combinations of these procedures. Screening of mRNA or genomic DNAmay be carried out with oligonucleotide probes generated from theglycosyltransferases gene sequence. Probes may be labeled with adetectable group such as a fluorescent group, a radioactive atom or achemiluminescent group in accordance with known procedures and used inconventional hybridization assays. In the alternative,glycosyltransferases gene sequences may be obtained by use of thepolymerase chain reaction (PCR) procedure, with the PCR oligonucleotideprimers being produced from the glycosyltransferases gene sequence. See,U.S. Pat. No. 4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 toMullis.

The glycosyltransferase may be synthesized in host cells transformedwith vectors containing DNA encoding the glycosyltransferases enzyme.Vectors are used either to amplify DNA encoding the glycosyltransferasesenzyme and/or to express DNA which encodes the glycosyltransferasesenzyme. An expression vector is a replicable DNA construct in which aDNA sequence encoding the glycosyltransferases enzyme is operably linkedto suitable control sequences capable of effecting the expression of theglycosyltransferases enzyme in a suitable host. The need for suchcontrol sequences will vary depending upon the host selected and thetransformation method chosen. Generally, control sequences include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation. Amplification vectors do not require expression controldomains. All that is needed is the ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants.

In an exemplary embodiment, the invention utilizes a prokaryotic enzyme.Such glycosyltransferases include enzymes involved in synthesis oflipooligosaccharides (LOS), which are produced by many gram negativebacteria (Preston et al., Critical Reviews in Microbiology 23(3):139-180 (1996)). Such enzymes include, but are not limited to, theproteins of the rfa operons of species such as E. coli and Salmonellatyphimurium, which include a β1,6 galactosyltransferase and a β1,3galactosyltransferase (see, e.g., EMBL Accession Nos. M80599 and M86935(E. coli); EMBL Accession No. S56361 (S. typhimurium)), aglucosyltransferase (Swiss-Prot Accession No. P25740 (E. coli), anβ1,2-glucosyltransferase (rfaJ)(Swiss-Prot Accession No. P27129 (E.coli) and Swiss-Prot Accession No. P19817 (S. typhimurium)), and anβ1,2-N-acetylglucosaminyltransferase (rfaK)(EMBL Accession No. U00039(E. coli). Other glycosyltransferases for which amino acid sequences areknown include those that are encoded by operons such as rfaB, which havebeen characterized in organisms such as Klebsiella pneumoniae, E. coli,Salmonella typhimurium, Salmonella enterica, Yersinia enterocolitica,Mycobacterium leprosum, and the rh1 operon of Pseudomonas aeruginosa.

Also suitable for use in the present invention are glycosyltransferasesthat are involved in producing structures containinglacto-N-neotetraose,D-galactosyl-β-1,4-N-acetyl-D-glucosaminyl-β-1,3-D-galactosyl-β-1,4-D-glucose,and the P^(k) blood group trisaccharide sequence,D-galactosyl-α-1,4-D-galactosyl-β-1,4-D-glucose, which have beenidentified in the LOS of the mucosal pathogens Neisseria gonnorhoeae andN. meningitidis (Scholten et al., J. Med. Microbiol. 41: 236-243(1994)). The genes from N. meningitidis and N. gonorrhoeae that encodethe glycosyltransferases involved in the biosynthesis of thesestructures have been identified from N. meningitidis immunotypes L3 andL1 (Jennings et al., Mol. Microbiol. 18: 729-740 (1995)) and the N.gonorrhoeae mutant F62 (Gotshlich, J. Exp. Med. 180: 2181-2190 (1994)).In N. meningitidis, a locus consisting of three genes, IgtA, IgtB and IgE, encodes the glycosyltransferase enzymes required for addition of thelast three of the sugars in the lacto-N-neotetraose chain (Wakarchuk etal., J. Biol. Chem. 271: 19166-73 (1996)). Recently the enzymaticactivity of the IgtB and IgtA gene product was demonstrated, providingthe first direct evidence for their proposed glycosyltransferasefunction (Wakarchuk et al., J. Biol. Chem. 271(45): 28271-276 (1996)).In N. gonorrhoeae, there are two additional genes, IgtD which addsβ-D-GalNAc to the 3 position of the terminal galactose of thelacto-N-neotetraose structure and IgtC which adds a terminal α-D-Gal tothe lactose element of a truncated LOS, thus creating the P^(k) bloodgroup antigen structure (Gotshlich (1994), supra.). In N. meningitidis,a separate immunotype L1 also expresses the P^(k) blood group antigenand has been shown to carry an IgtC gene (Jennings et al., (1995),supra.). Neisseria glycosyltransferases and associated genes are alsodescribed in U.S. Pat. No. 5,545,553 (Gotschlich). Genes forα1,2-fucosyltransferase and α1,3-fucosyltransferase from Helicobacterpylori has also been characterized (Martin et al., J. Biol. Chem. 272:21349-21356 (1997)). Also of use in the present invention are theglycosyltransferases of Campylobacter jejuni (see, for example,http://afmb.cnrs-mrs.fr/˜pedro/CAZY/gtf_(—)42.html).

a) Fucosyltransferases

In some embodiments, a glycosyltransferase used in the method of theinvention is a fucosyltransferase. Fucosyltransferases are known tothose of skill in the art. Exemplary fucosyltransferases includeenzymes, which transfer L-fucose from GDP-fucose to a hydroxy positionof an acceptor sugar. Fucosyltransferases that transfer non-nucleotidesugars to an acceptor are also of use in the present invention.

In some embodiments, the acceptor sugar is, for example, the GlcNAc in aGalβ(1→3,4)GlcNAcβ-group in an oligosaccharide glycoside. Suitablefucosyltransferases for this reaction include theGalβ(1→13,4)GlcNAcβ1-α(1→3,4)fucosyltransferase (FTIII E.C. No.2.4.1.65), which was first characterized from human milk (see, Palcic,et al., Carbohydrate Res. 190:1-11 (1989); Prieels, et al., J. Biol.Chem. 256: 10456-10463 (1981); and Nunez, et al., Can. J. Chem. 59:2086-2095 (1981)) and the Galβ(1→4)GlcNAcβ-αfucosyltransferases (FTIV,FTV, FTVI) which are found in human serum. FTVII (E.C. No. 2.4.1.65), asialyl α(2→3)Galβ((1→3)GlcNAcβ fucosyltransferase, has also beencharacterized. A recombinant form of the Galβ(1→3,4)GlcNAcβ-α(1→3,4)fucosyltransferase has also been characterized (see,Dumas, et al., Bioorg. Med. Letters 1: 425-428 (1991) andKukowska-Latallo, et al., Genes and Development 4: 1288-1303 (1990)).Other exemplary fucosyltransferases include, for example, α1,2fucosyltransferase (E.C. No. 2.4.1.69). Enzymatic fucosylation can becarried out by the methods described in Mollicone, et al., Eur. J.Biochem. 191: 169-176 (1990) or U.S. Pat. No. 5,374,655. Cells that areused to produce a fucosyltransferase will also include an enzymaticsystem for synthesizing GDP-fucose.

b) Galactosyltransferases

In another group of embodiments, the glycosyltransferase is agalactosyltransferase. Exemplary galactosyltransferases include α(1,3)galactosyltransferases (E.C. No. 2.4.1.151, see, e.g., Dabkowski et al.,Transplant Proc. 25:2921 (1993) and Yamamoto et al. Nature 345: 229-233(1990), bovine (GenBank j04989, Joziasse et al., J. Biol. Chem. 264:14290-14297 (1989)), murine (GenBank m26925; Larsen et al., Proc. Nat'l.Acad. Sci. USA 86: 8227-8231 (1989)), porcine (GenBank L36152; Strahanet al., Immunogenetics 41: 101-105 (1995)). Another suitable α1,3galactosyltransferase is that which is involved in synthesis of theblood group B antigen (EC 2.4.1.37, Yamamoto et al., J. Biol. Chem. 265:1146-1151 (1990) (human)). Yet a further exemplary galactosyltransferaseis core Gal-TI.

Also suitable for use in the methods of the invention are β(1,4)galactosyltransferases, which include, for example, EC 2.4.1.90 (LacNAcsynthetase) and EC 2.4.1.22 (lactose synthetase) (bovine (D'Agostaro etal., Eur. J. Biochem. 183: 211-217 (1989)), human (Masri et al.,Biochem. Biophys. Res. Commun. 157: 657-663 (1988)), murine (Nakazawa etal., J. Biochem. 104: 165-168 (1988)), as well as E.C. 2.4.1.38 and theceramide galactosyltransferase (EC 2.4.1.45, Stahl et al., J. Neurosci.Res. 38: 234-242 (1994)). Other suitable galactosyltransferases include,for example, α1,2 galactosyltransferases (from e.g., Schizosaccharomycespombe, Chapell et al., Mol. Biol. Cell 5: 519-528 (1994)).

c) Sialyltransferases

Sialyltransferases are another type of glycosyltransferase that isuseful in the recombinant cells and reaction mixtures of the invention.Cells that produce recombinant sialyltransferases will also produceCMP-sialic acid, which is a sialic acid donor for sialyltransferases.Examples of sialyltransferases that are suitable for use in the presentinvention include ST3Gal III (e.g., a rat or human ST3Gal III), ST3GalIV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II,and ST6GalNAc III (the sialyltransferase nomenclature used herein is asdescribed in Tsuji et al., Glycobiology 6: v-xiv (1996)). An exemplaryα(2,3)sialyltransferase referred to as α(2,3)sialyltransferase (EC2.4.99.6) transfers sialic acid to the non-reducing terminal Gal of aGalβ1→3Glc disaccharide or glycoside. See, Van den Eijnden et al., J.Biol. Chem. 256: 3159 (1981), Weinstein et al., J. Biol. Chem. 257:13845 (1982) and Wen et al., J. Biol. Chem. 267: 21011 (1992). Anotherexemplary α2,3-sialyltransferase (EC 2.4.99.4) transfers sialic acid tothe non-reducing terminal Gal of the disaccharide or glycoside. see,Rearick et al., J. Biol. Chem. 254: 4444 (1979) and Gillespie et al., J.Biol. Chem. 267: 21004 (1992). Further exemplary enzymes includeGal-β-1,4-GlcNAc α-2,6 sialyltransferase (See, Kurosawa et al. Eur. J.Biochem. 219: 375-381 (1994)).

Preferably, for glycosylation of carbohydrates of glycopeptides thesialyltransferase will be able to transfer sialic acid to the sequenceGalβ1,4GlcNAc-, the most common penultimate sequence underlying theterminal sialic acid on fully sialylated carbohydrate structures (see,Table 5).

TABLE 5 Sialyltransferases which use the Galβ1,4GlcNAc sequence as anacceptor substrate Sialyltransferase Source Sequence(s) formed Ref.ST6Gal I Mammalian NeuAcI2,6Galβ1,4GlCNAc- 1 ST3Gal III MammalianNeuAcI2,3Galβ1,4GlCNAc- 1 NeuAcI2,3Galβ1,3GlCNAc- ST3Gal IV MammalianNeuAcI2,3Galβ1,4GlCNAc- 1 NeuAcI2,3Galβ1,3GlCNAc- ST6Gal II MammalianNeuAcI2,6Galβ1,4GlCNA ST6Gal II photobacterium NeuAcI2,6Galβ1,4GlCNAc- 2ST3Gal V N. meningitides NeuAcI2,3Galβ1,4GlCNAc- 3 N. gonorrhoeae 1Goochee et al., Bio/Technology 9: 1347-1355 (1991) 2 Yamamoto et al., J.Biochem. 120: 104-110 (1996) 3 Gilbert et al., J. Biol. Chem. 271:28271-28276 (1996)

An example of a sialyltransferase that is useful in the claimed methodsis ST3Gal III, which is also referred to as α(2,3)sialyltransferase (EC2.4.99.6). This enzyme catalyzes the transfer of sialic acid to the Galof a Galβ1,3GlcNAc or Galβ1,4GlcNAc glycoside (see, e.g., Wen et al., J.Biol. Chem. 267: 21011 (1992); Van den Eijnden et al., J. Biol. Chem.256: 3159 (1991)) and is responsible for sialylation ofasparagine-linked oligosaccharides in glycopeptides. The sialic acid islinked to a Gal with the formation of an α-linkage between the twosaccharides. Bonding (linkage) between the saccharides is between the2-position of NeuAc and the 3-position of Gal. This particular enzymecan be isolated from rat liver (Weinstein et al., J. Biol. Chem. 257:13845 (1982)); the human cDNA (Sasaki et al. (1993) J. Biol. Chem. 268:22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem. 269: 1394-1401)and genomic (Kitagawa et al. (1996) J. Biol. Chem. 271: 931-938) DNAsequences are known, facilitating production of this enzyme byrecombinant expression. In a preferred embodiment, the claimedsialylation methods use a rat ST3Gal III.

Other exemplary sialyltransferases of use in the present inventioninclude those isolated from Campylobacter jejuni, including the α(2,3).See, e.g, WO99/49051.

Sialyltransferases other those listed in Table 5, are also useful in aneconomic and efficient large-scale process for sialylation ofcommercially important glycopeptides. As a simple test to find out theutility of these other enzymes, various amounts of each enzyme (1-100mU/mg protein) are reacted with asialo-α₁ AGP (at 1-10 mg/ml) to comparethe ability of the sialyltransferase of interest to sialylateglycopeptides relative to either bovine ST6Gal I, ST3Gal III or bothsialyltransferases. Alternatively, other glycopeptides, or N-linkedoligosaccharides enzymatically released from the peptide backbone can beused in place of asialo-al AGP for this evaluation. Sialyltransferaseswith the ability to sialylate N-linked oligosaccharides of glycopeptidesmore efficiently than ST6Gal I are useful in a practical large-scaleprocess for peptide sialylation (as illustrated for ST3Gal III in thisdisclosure).

d) GalNAc Transferases

N-acetylgalactosaminyltransferases are of use in practicing the presentinvention, particularly for binding a GalNAc moiety to an amino acid ofthe O-linked glycosylation site of the peptide. SuitableN-acetylgalactosaminyltransferases include, but are not limited to,α(1,3) N-acetylgalactosaminyltransferase, β(1,4)N-acetylgalactosaminyltransferases (Nagata et al., J. Biol. Chem. 267:12082-12089 (1992) and Smith et al., J Biol. Chem. 269:15162 (1994)) andpolypeptide N-acetylgalactosaminyltransferase (Homa et al., J. Biol.Chem. 268: 12609 (1993)).

Production of proteins such as the enzyme GalNAc T_(I-XX) from clonedgenes by genetic engineering is well known. See, eg., U.S. Pat. No.4,761,371. One method involves collection of sufficient samples, thenthe amino acid sequence of the enzyme is determined by N-terminalsequencing. This information is then used to isolate a cDNA cloneencoding a full-length (membrane bound) transferase which uponexpression in the insect cell line Sf9 resulted in the synthesis of afully active enzyme. The acceptor specificity of the enzyme is thendetermined using a semiquantitative analysis of the amino acidssurrounding known glycosylation sites in 16 different proteins followedby in vitro glycosylation studies of synthetic peptides. This work hasdemonstrated that certain amino acid residues are overrepresented inglycosylated peptide segments and that residues in specific positionssurrounding glycosylated serine and threonine residues may have a moremarked influence on acceptor efficiency than other amino acid moieties.

2. Sulfotransferases

The invention also provides methods for producing peptides that includesulfated molecules, including, for example sulfated polysaccharides suchas heparin, heparan sulfate, carragenen, and related compounds. Suitablesulfotransferases include, for example, chondroitin-6-sulphotransferase(chicken cDNA described by Fukuta et al., J. Biol. Chem. 270:18575-18580 (1995); GenBank Accession No. D49915), glycosaminoglycanN-acetylglucosamine N-deacetylase/N-sulphotransferase 1 (Dixon et al.,Genomics 26: 239-241 (1995); UL 18918), and glycosaminoglycanN-acetylglucosamine N-deacetylase/N-sulphotransferase 2 (murine cDNAdescribed in Orellana et al., J. Biol. Chem. 269: 2270-2276 (1994) andEriksson et al., J. Biol. Chem. 269: 10438-10443 (1994); human cDNAdescribed in GenBank Accession No. U2304).

3. Cell-Bound Glycosyltransferases

In another embodiment, the enzymes utilized in the method of theinvention are cell-bound glycosyltransferases. Although many solubleglycosyltransferases are known (see, for example, U.S. Pat. No.5,032,519), glycosyltransferases are generally in membrane-bound formwhen associated with cells. Many of the membrane-bound enzymes studiedthus far are considered to be intrinsic proteins; that is, they are notreleased from the membranes by sonication and require detergents forsolubilization. Surface glycosyltransferases have been identified on thesurfaces of vertebrate and invertebrate cells, and it has also beenrecognized that these surface transferases maintain catalytic activityunder physiological conditions. However, the more recognized function ofcell surface glycosyltransferases is for intercellular recognition(Roth, MOLECULAR APPROACHES to SUPRACELLULAR PHENOMENA, 1990).

Methods have been developed to alter the glycosyltransferases expressedby cells. For example, Larsen et al., Proc. Natl. Acad. Sci. USA 86:8227-8231 (1989), report a genetic approach to isolate cloned cDNAsequences that determine expression of cell surface oligosaccharidestructures and their cognate glycosyltransferases. A cDNA librarygenerated from mRNA isolated from a murine cell line known to expressUDP-galactose:.β.-D-galactosyl-1,4-N-acetyl-D-glucosaminideα-1,3-galactosyltransferase was transfected into COS-1 cells. Thetransfected cells were then cultured and assayed for α 1-3galactosyltransferase activity.

Francisco et al., Proc. Natl. Acad. Sci. USA 89: 2713-2717 (1992),disclose a method of anchoring β-lactamase to the external surface ofEscherichia coli. A tripartite fusion consisting of (i) a signalsequence of an outer membrane protein, (ii) a membrane-spanning sectionof an outer membrane protein, and (iii) a complete mature β-lactamasesequence is produced resulting in an active surface bound β-lactamasemolecule. However, the Francisco method is limited only to procaryoticcell systems and as recognized by the authors, requires the completetripartite fusion for proper functioning.

4. Fusion Proteins

In other exemplary embodiments, the methods of the invention utilizefusion proteins that have more than one enzymatic activity that isinvolved in synthesis of a desired glycopeptide conjugate. The fusionpolypeptides can be composed of, for example, a catalytically activedomain of a glycosyltransferase that is joined to a catalytically activedomain of an accessory enzyme. The accessory enzyme catalytic domaincan, for example, catalyze a step in the formation of a nucleotide sugarthat is a donor for the glycosyltransferase, or catalyze a reactioninvolved in a glycosyltransferase cycle. For example, a polynucleotidethat encodes a glycosyltransferase can be joined, in-frame, to apolynucleotide that encodes an enzyme involved in nucleotide sugarsynthesis. The resulting fusion protein can then catalyze not only thesynthesis of the nucleotide sugar, but also the transfer of the sugarmoiety to the acceptor molecule. The fusion protein can be two or morecycle enzymes linked into one expressible nucleotide sequence. In otherembodiments the fusion protein includes the catalytically active domainsof two or more glycosyltransferases. See, for example, U.S. Pat. No.5,641,668. The modified glycopeptides of the present invention can bereadily designed and manufactured utilizing various suitable fusionproteins (see, for example, PCT Patent Application PCT/CA98/01180, whichwas published as WO 99/31224 on Jun. 24, 1999.)

5. Immobilized Enzymes

In addition to cell-bound enzymes, the present invention also providesfor the use of enzymes that are immobilized on a solid and/or solublesupport. In an exemplary embodiment, there is provided aglycosyltransferase that is conjugated to a PEG via an intact glycosyllinker according to the methods of the invention. The PEG-linker-enzymeconjugate is optionally attached to solid support. The use of solidsupported enzymes in the methods of the invention simplifies the work upof the reaction mixture and purification of the reaction product, andalso enables the facile recovery of the enzyme. The glycosyltransferaseconjugate is utilized in the methods of the invention. Othercombinations of enzymes and supports will be apparent to those of skillin the art.

Purification of Peptide Conjugates

The products produced by the above processes can be used withoutpurification. However, it is usually preferred to recover the product.Standard, well-known techniques for recovery of glycosylated saccharidessuch as thin or thick layer chromatography, column chromatography, ionexchange chromatography, or membrane filtration can be used. It ispreferred to use membrane filtration, more preferably utilizing areverse osmotic membrane, or one or more column chromatographictechniques for the recovery as is discussed hereinafter and in theliterature cited herein. For instance, membrane filtration wherein themembranes have molecular weight cutoff of about 3000 to about 10,000 canbe used to remove proteins such as glycosyl transferases. Nanofiltrationor reverse osmosis can then be used to remove salts and/or purify theproduct saccharides (see, e.g., WO 98/15581). Nanofilter membranes are aclass of reverse osmosis membranes that pass monovalent salts but retainpolyvalent salts and uncharged solutes larger than about 100 to about2,000 Daltons, depending upon the membrane used. Thus, in a typicalapplication, saccharides prepared by the methods of the presentinvention will be retained in the membrane and contaminating salts willpass through.

If the modified glycoprotein is produced intracellularly, as a firststep, the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration; optionally,the protein may be concentrated with a commercially available proteinconcentration filter, followed by separating the polypeptide variantfrom other impurities by one or more steps selected from immunoaffinitychromatography, ion-exchange column fractionation (e.g., ondiethylaminoethyl (DEAE) or matrices containing carboxymethyl orsulfopropyl groups), chromatography on Blue-Sepharose, CMBlue-Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharose, WGA-Sepharose,Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, Phenyl Toyopearl,SP-Sepharose, or protein A Sepharose, SDS-PAGE chromatography, silicachromatography, chromatofocusing, reverse phase HPLC (e.g., silica gelwith appended aliphatic groups), gel filtration using, e.g., Sephadexmolecular sieve or size-exclusion chromatography, chromatography oncolumns that selectively bind the polypeptide, and ethanol or ammoniumsulfate precipitation.

Modified glycopeptides produced in culture are usually isolated byinitial extraction from cells, enzymes, etc., followed by one or moreconcentration, salting-out, aqueous ion-exchange, or size-exclusionchromatography steps, e.g., SP Sepharose. Additionally, the modifiedglycoprotein may be purified by affinity chromatography. HPLC may alsobe employed for one or more purification steps.

A protease inhibitor, e.g, methylsulfonylfluoride (PMSF) may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

Within another embodiment, supernatants from systems which produce themodified glycopeptide of the invention are first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate may be applied to a suitablepurification matrix. For example, a suitable affinity matrix maycomprise a ligand for the peptide, a lectin or antibody molecule boundto a suitable support. Alternatively, an anion-exchange resin may beemployed, for example, a matrix or substrate having pendant DEAE groups.Suitable matrices include acrylamide, agarose, dextran, cellulose, orother types commonly employed in protein purification. Alternatively, acation-exchange step may be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are particularly preferred.

Finally, one or more RP-HPLC steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, may beemployed to further purify a polypeptide variant composition. Some orall of the foregoing purification steps, in various combinations, canalso be employed to provide a homogeneous modified glycoprotein.

The modified glycopeptide of the invention resulting from a large-scalefermentation may be purified by methods analogous to those disclosed byUrdal et al., J Chromatog. 296: 171 (1984). This reference describes twosequential, RP-HPLC steps for purification of recombinant human IL-2 ona preparative HPLC column. Alternatively, techniques such as affinitychromatography may be utilized to purify the modified glycoprotein.

Pharmaceutical Compositions

Polypeptides modified at various O-linked glycosylation site accordingto the method of the present invention have a broad range ofpharmaceutical applications. For example, GLP-1 may be used for thetreatment or prevention of diabetes or obesity.

An additional example, human growth hormone (hGH) modified according tothe methods of the present invention may be used to treat growth-relatedconditions such as dwarfism, short-stature in children and adults,cachexia/muscle wasting, general muscular atrophy, and sex chromosomeabnormality (e.g., Turner's Syndrome). Other conditions may be treatedusing modified hGH include: short-bowel syndrome, lipodystrophy,osteoporosis, uraemaia, burns, female infertility, bone regeneration,general diabetes, type II diabetes, osteo-arthritis, chronic obstructivepulmonary disease (COPD), and insomia. Moreover, modified hGH may alsobe used to promote various processes, e.g., general tissue regeneration,bone regeneration, and wound healing, or as a vaccine adjunct.

Thus, in one aspect, the invention provides a pharmaceuticalcomposition. The pharmaceutical composition includes a pharmaceuticallyacceptable diluent and a covalent conjugate between anon-naturally-occurring, water-soluble polymer, therapeutic moiety orbiomolecule and a glycosylated or non-glycosylated peptide. The polymer,therapeutic moiety or biomolecule is conjugated to the peptide via anintact glycosyl linking group interposed between and covalently linkedto both the peptide and the polymer, therapeutic moiety or biomolecule.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

The pharmaceutical compositions may be formulated for any appropriatemanner of administration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablematrises, such as microspheres (e.g., polylactate polyglycolate), mayalso be employed as carriers for the pharmaceutical compositions of thisinvention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

Commonly, the pharmaceutical compositions are administeredsubcutaneously or parenterally, e.g., intravenously. Thus, the inventionprovides compositions for parenteral administration which comprise thecompound dissolved or suspended in an acceptable carrier, preferably anaqueous carrier, e.g., water, buffered water, saline, PBS and the like.The compositions may also contain detergents such as Tween 20 and Tween80; stablizers such as mannitol, sorbitol, sucrose, and trehalose; andpreservatives such as EDTA and m-cresol. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably from 5 to 9 and most preferably from 7 and 8.

In some embodiments the glycopeptides of the invention can beincorporated into liposomes formed from standard vesicle-forming lipids.A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of liposomesusing a variety of targeting agents (e.g., the sialyl galactosides ofthe invention) is well known in the art (see, e.g., U.S. Pat. Nos.4,957,773 and 4,603,044).

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes of lipidcomponents, such as phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid-derivatized glycopeptides of the invention.

Targeting mechanisms generally require that the targeting agents bepositioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. The carbohydrates of the invention maybe attached to a lipid molecule before the liposome is formed usingmethods known to those of skill in the art (e.g., alkylation oracylation of a hydroxyl group present on the carbohydrate with a longchain alkyl halide or with a fatty acid, respectively). Alternatively,the liposome may be fashioned in such a way that a connector portion isfirst incorporated into the membrane at the time of forming themembrane. The connector portion must have a lipophilic portion, which isfirmly embedded and anchored in the membrane. It must also have areactive portion, which is chemically available on the aqueous surfaceof the liposome. The reactive portion is selected so that it will bechemically suitable to form a stable chemical bond with the targetingagent or carbohydrate, which is added later. In some cases it ispossible to attach the target agent to the connector molecule directly,but in most instances it is more suitable to use a third molecule to actas a chemical bridge, thus linking the connector molecule which is inthe membrane with the target agent or carbohydrate which is extended,three dimensionally, off of the vesicle surface.

The following examples are provided to illustrate the conjugates, andmethods and of the present invention, but not to limit the claimedinvention.

EXAMPLES Example 1 1.1 Preparation of Glucagon-Like Peptide MutantsComprising Artificial Glycosylation Sites

Mutations in the amino acid sequence of Glucagon-Like Peptide-1 (GLP-1)will be made in order to introduce sites for O-linked glycosylation,such that the protein may be modified at these sites using the method ofthe present invention. Mutatants can be created using well known methodsfor solid state synthesis. Alternatively, mutations will be introducedinto a nucleic acid the sequence encoding GLP-1 such that O-linkedglycosylation sites will be introduced at each position along thepeptide back bone.

The following are some exemplary GLP-1 mutants.

GLP-1 Glycopeptides

Ac-X-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂H-X-EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂HA-X-GTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂HAE-X-TFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂HAEG-X-FTSDVSSYLEGQAAKEFIAWLVKGR-NH₂HAEGT-X-TSDVSSYLEGQAAKEFIAWLVKGR-NH₂HAEGTF-X-SDVSSYLEGQAAKEFIAWLVKGR-NH₂HAEGTFT-X-DVSSYLEGQAAKEFIAWLVKGR-NH₂HAEGTFTS-X-VSSYLEGQAAKEFIAWLVKGR-NH₂HAEGTFTSD-X-SSYLEGQAAKEFIAWLVKGR-NH₂HAEGTFVSDV-X-SYLEGQAAKEFIAWLVKGR-NH₂HAEGTFTSDVS-X-YLEGQAAKEFIAWLVKGR-NH₂HAEGTFTSDVSS-X-LEGQAAKEFIAWLVKGR-NH₂HAEGTFTSDVSSY-X-EGQAAKEFIAWLVKGR-NH₂HAEGTFTSDVSSYL-X-GQAAKEFIAWLVKGR-NH₂HAEGTFTSDVSSYLE-X-QAAKEFIAWLVKGR-NH₂HAEGTFTSDVSSYLEG-X-AAKEFIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQ-X-AKEFIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQA-X-KEFIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQAA-X-EFIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQAAK-X-FIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQAAKE-X-IAWLVKGR-NH₂HAEGTFTSDVSSYLEGQAAKEF-X-AWLVKGR-NH₂HAEGTFTSDVSSYLEGQAAKEFI-X-WLVKGR-NH₂HAEGTFTSDVSSYLEGQAAKEFIA-X-LVKGR-NH₂HAEGTFTSDVSSYLEGQAAKEFIAW-X-VKGR-NH₂HAEGTFTSDVSSYLEGQAAKEFIAWL-X-KGR-NH₂HAEGTFTSDVSSYLEGQAAKEFIAWLV-X-GR-NH₂HAEGTFTSDVSSYLEGQAAKEFIAWLVK-X-R-NH₂ HAEGTFTSDVSSYLEGQAAKEFIAWLVKG-X-NH₂HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-X-NH₂

1.2 Preparation of GLP-1-GalNAc (pH 6.2)

GLP-1 (960 μg) in 3.2 mL of buffer will be concentrated byutrafiltration using an UF filter (5 KDa) and then reconstituted with 1mL of 25 mM MES buffer (pH 6.2, 0.005% NaN₃). The UDP-GalNAc (6 mg, 9.24mM), GalNAc-T2 (40 μL, 0.04 U), and 100 mM MnCl₂ (40 μL, 4 mM) will thenbe added and the resulting solution will be incubated at roomtemperature. After 48 h, the MALDI should the reaction was complete(shift of the mass ion from 18800 to 19023 mass units). The reactionmixture will be purified by HPLC using SEC (Superdex 75 and Superdex200). The column will be eluted using phosphate buffered saline, pH 4.9and 0.005% tween 80. The peak corresponding to GLP-1-GalNAc will becollected and concentrated to about 150 μL using a Centricon 5 KDafilter and the volume will be adjusted to 1 mL using PBS (phosphatebuffered saline, pH 4.9 and 0.005% tween 80); protein concentration 1mg/mL A₂₈₀).

1.3 Preparation of GLP-1-GalNAc-Gal (pH 6.0)

GLP-1-GalNAc (100 μg) will be added to a 100 μL of a solution containing25 mM MES buffer, pH 6.0, 1.5 mM UDP-GalNAc, 10 mM MgCl₂ and 80 mUGalNAc-T2. The CMP-SA-PEG-20 KDa (0.5 mg, 0.025 μmole), UDP-galactose 75μg (0.125 μmole), core-1-Gal-T 20 μL (10 mU) will then be added and thesolution slowly rocked at 32° C. for 24 h. MALDI should indicatecomplete conversion of GLP-1-GalNAc into GLP-1-GalNAc-Gal.

1.4 Preparation of GLP-1-F-GalNAc-SA-PEG-20 KDa (C)

1.3a Sequential Process (pH 6.2).

A GLP-1-GalNAc solution containing 1 mg of protein will be bufferexchanged into 25 mM MES buffer (pH 6.2, 0.005% NaN₃) and CMP-SA-PEG (20KDa) (5 mg, 0.25 mmole). MnCl₂ (100 μL, 100 mM solution) and ST6GalNAc-I(100 μL) will be added and the reaction mixture will be rocked slowly at32° C. Aliquots will be taken at time points (24, 48 and 72 h) andanalyzed by SDS-PAGE. After 24 h, no further reaction should beobserved. The reaction mixture will be concentrated by spin filtration(5 KDa), buffer exchanged with 25 mM NaOAc (pH 4.9) and concentrated to1 mL. The product will bes purified using ion exchange (SP-Sepharose, 25mM NaOAc, pH 4.9) and SEC (Superdex 75; PBS-pH 7.2, 0.005% tween 80, 1ml/min). The desired fraction will be collected, concentrated to 0.5 mLand stored at 4° C.

1.4b One Pot Process using ST6GalNAc-I (pH 6.0)

GLP-1 (960 μg of protein dissolved in 3.2 mL of product formulationbuffer) will be concentrated by spin filtration (5 KDa) to 0.5 mL andreconstituted in 25 mM MES buffer (pH 6.0, 0.005% NaN₃) to a totalvolume of about 1 mL, or a protein concentration of 1 mg/mL. UDP-GalNAc(6 mg, 9.21 μmol), GalNAc-T2 (80 μL, 80 mU), CMP-SA-PEG (20 KDa) (6 mg,0.3 μmol) and mouse enzyme ST6GalNAc-I (120 μL will be added). Thesolution will be rocked at 32° C. for 48 h and purified using standardchromatography conditions on SP-Sepharose and SEC as described above. Atotal of 0.5 mg of protein (A₂₈₀) should be obtained, or about a 50%overall yield. The product structure will be confirmed by analysis withboth MALDI and SDS-PAGE.

1.5 Preparation of GLP-1-GalNAc-Gal-SA-PEG-20 KDa (D)

1.5a Starting from GLP-1-GalNAc

UDP-galactose (4 mg, 6.5 μmole), core-1-Gal-T₁ (320 μL, 160 mU),CMP-SA-PEG-20 KDa (8 mg, 0.4 μmole), ST3Gal2 (80 μL, 0.07 mU) and 100 mMMnCl₂ (80 μL) will be directly added to the crude reaction mixture ofthe GLP-1-GalNAc (1.5 mg) in 25 mM MES buffer (pH 6.0), 1.5 mL, asdescribed above. The resulting mixture will be incubated at 32° C. for60 h, however, the reaction should be complete after 24 h. The reactionmixture will be centrifuged and the solution was concentrated to 0.2 mLusing ultrafiltration (5 KDa) and then redissolved in 25 mM NaOAc (pH4.5) to a final volume of 1 mL. The product will be purified usingSP-Sepharose, the peak fractions were concentrated using a spin filter(5 KDa), and the residue purified further using SEC (Superdex 75). Afterconcentration using a spin filter (5 KDa), the protein will be dilutedto 1 mL using formulation buffer (PBS, 2.5% mannitol, 0.005%polysorbate, pH 6.5) and formulated at a protein concentration of 850 μgprotein per mL (A₂₈₀). The overall yield should be around 55%.

1.5b Starting from GLP-1

GLP-1 (960 μg, 3.2 mL) will be concentrated by spin filter (5 KDa) andreconstituted with 25 mM MES buffer (pH 6.0, 0.005% NaN₃). The totalvolume of the GLP-1 solution will be adjusted to about 1 mg/mL andUDP-GalNAc (6 mg), GalNAc-T2 (80 μL), UDP-galactose (6 mg),core-1-Gal-T₁ (160 μL, 80 μU), CMP-SA-PEG (20 KDa) (6 mg), ST3Gal-2 (160μL, 120 μU) and MnCl₂ (40 μL of a 100 mM solution) will be added. Theresulting mixture will be incubated at 32° C. for 48 h.

1.6 SP Sepharose HPLC Chromatography

The SP Sepharose column (HiTrap HP, FF, 1 mL, Amersham) can be used witha Varian HPLC system to separate individual GLP-1 peptides from crudeextracts. Absorbance at 280 nm will be monitored. The column will bewashed with 20 mL of 2 M NaCl in 25 mM sodium acetate (pH 4.5) contained0.005% polysorbate 80 and was equilibrated with 20 mL of 25 mM sodiumacetate (pH 4.5) contained 0.005% polysorbate 80 at a flow rate of 1.0mL/min. The sample (about 0.5 mg/200 μL) will be injected onto thecolumn and the product will be eluted using the gradient: 0-10 min, 25mM NaAc, pH 4.5, 0.005% Polysorbate 80; 10-20 min, a gradient of 0-0.5 MNaCl in 25 mM NaAc, pH 4.5, 0.005% Polysorbate 80; 20-25 min, a gradientof 0.5 M-0.0 M NaCl in 25 mM NaAc, pH 4.5, 0.005% Polysorbate 80; and25-30 min, 25 mM NaAc, pH 4.5, 0.005% Polysorbate 80). Fractions will becollected and concentrated to about 1 mL by using 5 KDa filter foranalysis and further purification. Samples will be stored at 4° C.

1.6 Size Exclusion Chromatography

A Varian HPLC system containing a Superdex 75 column (HR 10/30, 10×300mm, Amersham) will be used at a flow rate of 1.0 mL/min, whilemonitoring absorbance at 280 nm. The sample will be injected (about 0.2mg/200 μL) and eluted with PBS, pH 7.4, 0.005% Polysorbate 80. Fractionswill be collected and concentrated to about 1 mL by using a 5 KDafilter. Samples will be stored at 4° C.

1.7 SDS PAGE Analysis

4-20% acrylamide gradient slab gels will be used. Samples will be mixedwith SDS Sample Buffer contained 1 mM DTT, and heated at 85° C. for 6min. Samples will be run in gel under a consistent voltage at 125 mV for1 h 50 min. After electrophoresis, the proteins will be stained withcolloidal stain solution at room temperature for 2-24 hours depended onthe protein concentration. The standard proteins shown on Tris-Glycinegel will be myosin (250 KDa), phosphorylase (148 KDa), BSA (98 KDa,glutamic dehydrogenase (64 KDa), alcohol dehydrogenase (50 KDa),carbonic anhydrase (36 KDa), lysozyme (22 KDa), aprotinin (6 KDa), andinsulin β-chain (4 KDa). The protein bands in wet gel will be visualizedusing an HP Scanjet 7400C, and the picture of gel will be optimizedusing the HP Precision Scan Program.

1.8 MALDI Analysis

Samples will be dialyzed for 45 min using an MF-Millipore membranefilter (0.025 μm pore, 47 mm dia), floating on water. The dialyzedaliquots will be dried on a speedvac, re-dissolved in a small amount ofwater, and mixed with a solution of 2,5-dihydroxybenzoic acid (9 g/L)and 5-methoxysalicylic acid (1 g/L) dissolved in water/acetonitrile(50:50). The mixtures will be dried onto the MALDI target and analyzedusing an Applied Biosystems DE-Pro mass spectrometer operated in thelinear/negative-ion mode (Analytic lab, Neose Tech., Horsham, Pa.).

1.9 Peptide Mapping Analysis

Protein sample will be digested by trypsin overnight at 37° C. andloaded on a LC-MS system equipped with a Finnigan LCQ-classic ion trapmass spectrometer system with a electrospray ion source interfaced to a15 cm×300 um id LC Packings PepMap reverse-phase capillarychromatography column. 1 μL volume of the extract will be injected andthe peptides will be eluted from the column using a CH₃CN/0.1% formicacid gradient at a flow rate of 3 μL/min. The electrospray ion sourcewill be operated at 4.0 kV. The digest will be analyzed using the datadependent multitask capability of the instrument acquiring full scanmass spectra to determine peptide molecular weights and product ionspectra to determine amino acid sequence in successive instrument scans.This mode of analysis produced approximately 1100 collisionally induceddissociation (CID) spectra of ions ranging in abundance over severalorders of magnitude.

The data will be analyzed by locating the ten to fifteen most abundantions in a base peak presentation of the full scan data and interpretingthe CID spectra of those ions to produce the tabulated results for eachdigest.

1.10 Protein Concentration Assay

Protein concentration will be determined by spectrophotometer at a fixedabsorbance of 280 nm with 1 cm path length of cell. Triplicate readingswill be measured for a tested sample with water and buffer as controls.Protein concentration will be determined using extinction coefficient at0.799 mL/mg protein.

1.11 Formulation of Final Product

The formulation buffer contained pyrogen-free PBS, pH 6.5, 2.5%mannitol, and 0.05% Polysorbate 80 that will be degassed by vacuum andsterile filtered (0.2 μm).

Any endotoxin will be removed using a Detoxi-Gel™ equilibrated with 5column beds of the formulation buffer (PBS, pH 6.5, 2.5% mannitol, and0.05% Polysorbate 80). The flow rate was controlled by gravity at ˜0.3mL/min. Product samples will be applied onto the gel, and the producteluted using the formulation buffer. The volume of the collected productwill be adjusted with additional formulation buffer to provide a proteinconcentration of about 100 μg/mL.

The peptide formulations will be sterile filtered (0.2μ) and theeffluent will be dispensed as 1 mL aliquots into 2.0 mL pyrogen-freevials. In addition, aliquots will be taken for endotoxin and proteinanalysis. All products will be stored at 4° C.

1.12 Endotoxin Determination

Endotoxin contamination will be determined using Limulus AmebocyteLysate (LAL) assay (BioWhittaker, Kinetic-QCL Kit, Cat#: 50-650U).

Example 2 Determination of Biological Activity of GLP-1 Peptides

Methods for measuring biological activity of GLP-1 are known in the art.In particular, GLP-1 activity will be measured in vivo and in vitro asdisclosed in Xiao Q., et al. (2001) Biochemistry 40:2860-2869, which isincorporated herein by reference in its entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

All patents, patent applications, and other publications cited in thisapplication are incorporated by reference in the entirety.

1. A peptide having a formula selected from:

in which AA is an amino acid with a side chain that comprises a hydroxylmoiety; and X a modifying group or it is a saccharyl moiety.
 2. Thepeptide according to claim 1, wherein AA is introduced into said peptidevia mutation of wild-type peptide.
 3. The peptide according to claim 1,wherein X comprises a group selected from sialyl, galactosyl and Gal-Siamoieties, wherein at least one of said sialyl, galactosyl and Gal-Siacomprises a modifying group.
 4. The peptide according to claim 1,wherein X comprises poly(ethylene glycol).
 5. The peptide according toclaim 1, wherein X comprises monomethoxy-poly(ethylene glycol).
 6. Thepeptide according to claim 5, wherein X comprises the structure:

in which L is a substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl group; and n is selected from the integersfrom 0 to about
 500. 7. The peptide according to claim 5, wherein Xcomprises the structure:

in which s is selected from the integers from 0 to
 20. 8. An isolatednucleic acid comprising a polynucleotide sequence encoding a mutantpolypeptide, wherein the mutant polypeptide comprises an O-linkedglycosylation site that does not exist in the corresponding wild-typepolypeptide.
 9. The nucleic acid of claim 8, wherein the polypeptide isa GLP-1 polypeptide.
 10. An expression cassette comprising the nucleicacid of claim
 8. 11. A cell comprising the nucleic acid of claim
 8. 12.A method for making a glycoconjugate of a mutant polypeptide, whichcomprises an O-linked glycosylation that does not exist in thecorresponding wild-type polypeptide, comprising the steps of: (a)recombinantly producing the mutant polypeptide, and (b) enzymaticallyglycosylating the mutant polypeptide with a modified sugar at saidO-linked glycosylation site.
 13. The method of claim 12, wherein thecorresponding mutant polypeptide has an amino acid sequence selectedfrom the group consisting of: GLP-1 Glycopeptides

Ac-X-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂H-X-EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂HA-X-GTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂HAE-X-TFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂HAEG-X-FTSDVSSYLEGQAAKEFIAWLVKGR-NH₂HAEGT-X-TSDVSSYLEGQAAKEFIAWLVKGR-NH₂HAEGTF-X-SDVSSYLEGQAAKEFIAWLVKGR-NH₂HAEGTFT-X-DVSSYLEGQAAKEFIAWLVKGR-NH₂HAEGTFTS-X-VSSYLEGQAAKEFIAWLVKGR-NH₂HAEGTFTSD-X-SSYLEGQAAKEFIAWLVKGR-NH₂HAEGTFVSDV-X-SYLEGQAAKEFIAWLVKGR-NH₂HAEGTFTSDVS-X-YLEGQAAKEFIAWLVKGR-NH₂HAEGTFTSDVSS-X-LEGQAAKEFIAWLVKGR-NH₂HAEGTFTSDVSSY-X-EGQAAKEFIAWLVKGR-NH₂HAEGTFTSDVSSYL-X-GQAAKEFIAWLVKGR-NH₂HAEGTFTSDVSSYLE-X-QAAKEFIAWLVKGR-NH₂HAEGTFTSDVSSYLEG-X-AAKEFIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQ-X-AKEFIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQA-X-KEFIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQAA-X-EFIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQAAK-X-FIAWLVKGR-NH₂HAEGTFTSDVSSYLEGQAAKE-X-IAWLVKGR-NH₂HAEGTFTSDVSSYLEGQAAKEF-X-AWLVKGR-NH₂HAEGTFTSDVSSYLEGQAAKEFI-X-WLVKGR-NH₂HAEGTFTSDVSSYLEGQAAKEFIA-X-LVKGR-NH₂HAEGTFTSDVSSYLEGQAAKEFIAW-X-VKGR-NH₂HAEGTFTSDVSSYLEGQAAKEFIAWL-X-KGR-NH₂HAEGTFTSDVSSYLEGQAAKEFIAWLV-X-GR-NH₂HAEGTFTSDVSSYLEGQAAKEFIAWLVK-X-R-NH₂ HAEGTFTSDVSSYLEGQAAKEFIAWLVKG-X-NH₂HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-X-NH₂


14. A pharmaceutical composition of a Glucagon-Like Peptide-1 comprisingan effective amount of a mutant polypeptide, which comprises an O-linkedglycosylation site that does not exist in the corresponding wild-typeGlucagon-Like Peptide-1, wherein said peptide is glycoconjugated with amodified sugar.
 15. The pharmaceutical composition according to claim13, wherein said modified sugar is modified with a member selected frompoly(ethylene glycol) and m-poly(ethylene glycol).
 16. A method ofproviding Glucagon-Like Peptide-1 therapy to a subject in need of saidtherapy, said method comprising, administering to said subject an amountof an O-linked glyco-PEG-ylated Glucagon-Like Peptide-1 sufficient toprovide a therapeutic effect.
 17. The method according to claim 16,wherein said O-linked glyco-PEG-ylated Glucagon-Like Peptide-1 isglyco-PEG-ylated on an amino acid residue not present in wild typeGlucagon-Like Peptide-1.